Upstream control elements of the proopiomelanocortin gene and their use

ABSTRACT

The nucleic acid sequence of the POMC enhancer is disclosed herein. Sequences from the human, rat, rabbit, hamster, mouse, and cow POMC enhancer are disclosed. Hybrid transgenes, comprising a POMC transcriptional control element operably linked to a nucleic acid sequence encoding a marker are also enclosed. In addition, transgenic mice carrying a hybrid transgene including a POMC control element operably linked to a marker are disclosed herein.

PRIORITY

This is a divisional of U.S. patent application Ser. No. 10/336,091filed Jan. 3, 2003, now U.S. Pat. No. 7,125,979 which is acontinuation-in-part of U.S. application Ser. No. 10/255,175, filed Sep.24, 2002 now abandoned, which claims the benefit of U.S. ProvisionalApplication No. 60/324,406, filed Sep. 24, 2001, and claims the benefitof U.S. Provisional Application No. 60/392,109, filed Jun. 28, 2002. Allof the prior applications are incorporated by reference in theirentirety herein.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States government support pursuantto grants TW01233 and DK55819, from the National Institutes of Health.The United States government has certain rights in the invention.

FIELD

This application relates to the field of transcriptional controlelements, specifically to enhancers of the proopiomelanocortin (POMC)gene, and the use of these elements to direct expression of aheterologous nucleic acid sequence. Transgenic mice carrying a transgenethat include transcriptional regulatory regions of a POMC gene are alsodisclosed.

BACKGROUND

POMC neurons of the hypothalamus are critical components of the neuralcircuitry controlling appetite, feeding, and metabolism. In additionthey modulate the activity of other hypothalamic neurons which controlreproduction, thyroid hormone levels, growth hormone secretion,prolactin secretion, and neuroendocrine stress responses. These neuronsalso are involved in the nervous system's endogenous analgesic andreward circuits. POMC is a prohormone that is postranslationallyprocessed into several different biologically active neuropeptides. Themost important of these neuropeptides within the hypothalamus arealpha-MSH, gamma-MSH, and the opioid beta-endorphin. The two MSHpeptides are potent anorexigenic substances that play a fundamental rolein modulating weight homeostasis. β-endorphin also modulates food intakeby at least two distinct mechanisms. It can directly increase foodintake when administered acutely, but it also modulates the neuralcircuitry underlying the rewarding aspects of food ingestion and therebyinfluences an organism's motivation to work to obtain food.

Genetic evidence in humans implicates the POMC gene in the regulation ofweight and fat mass. Rare mutations causing null alleles result in asyndrome of adrenal cortical insufficiency, red hair, and obesity. Thethree components of the syndrome are secondary to losses of peripheralACTH, peripheral MSH, and central MSH, respectively. However, a numberof other gene association and quantitative trait loci analyses in humanpopulations suggest a much more common role for the POMC gene in weighthomeostasis.

The POMC promoter has been identified. For example, in the rat, 5′flanking sequences form −323 to −34 are sufficient for correct spatial,temporal and hormonally regulated expression of POMC in the pituitarygland (e.g. see Liu et al., Mol. Cell. Biol. 12:3978-3990, 1992; Liu etal., Biochem. J. 312:827-832, 1995). In addition, a transgene includingapproximately 13 kilobases (kb) of the mouse POMC gene has beendemonstrated to produce cell specific and developmentally regulatedexpression of POMC in transgenic mice (Young et al., J. Neurosci.18:6631-6640, 1998). However, other specific nucleic acid elementsinvolved in tissue specific expression, such as a POMC enhancer, havenot been identified.

SUMMARY

The nucleic acid sequence of the transcriptional control region of theproopiomelanocortin (POMC) gene is disclosed herein. Specifically, ahuman, rat, rabbit, mouse, hamster, and cow POMC enhancer element aredescribed herein, and the use of these enhancer elements to directtranscription is disclosed. Hybrid transgenes, comprising a POMCtranscriptional control element operably linked to a nucleic acidsequence encoding a marker are also enclosed. In addition, transgenicmice carrying a hybrid transgene including a POMC control elementoperably linked to a marker are disclosed herein.

Specifically, an isolated POMC enhancer element is disclosed herein. Theelement has a nucleic acid sequence set forth as a) SEQ ID NO: 9 or atmost fifty conserved enhancer substitutions thereof; b) SEQ ID NO: 15 orat most twenty conserved enhancer substitutions thereof; or c) SEQ IDNO: 19 or at most fifteen conserved enhancer substitutions thereof.

A POMC enhancer element is disclosed herein that includes one or more ofa) an nPOMC1 element comprising a nucleic acid sequence as set forth asSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or a conserved enhancersubstitution thereof based on a template nucleic acid sequence as setforth as SEQ ID NO: 9; b) an nPOMC2 element comprising a nucleic acidsequence as set forth as SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, or a conserved enhancer substitutionthereof based on a template nucleic acid sequence as set forth as SEQ IDNO: 15; c) an nPOMC3 element comprising a nucleic acid sequence as setforth as SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or a conservedenhancer substitution thereof based on a template nucleic acid sequenceas set forth as SEQ ID NO: 19. These expression elements directexpression of a heterologous nucleic acid sequence in aproopiomelanocortin neuron. An isolated enhancer element including anucleic acid sequence at least 90% homologous to an enhancer elementcomprising a nucleic acid sequence set forth as SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, or SEQ ID NO: 19 is also disclosed. The enhancerelement directs expression of a heterologous nucleic acid sequence in aproopiomelanocortin neuron. Vectors including these enhancer elements,and host cells transfected with these vectors, are also disclosed.

A transgenic mouse is described that expresses a marker inproopiomelanocortin neurons. The mouse carries a transgene comprising anucleic acid encoding the marker operably linked to aproopiomelanocortin enhancer sequence. The proopiomelanocortin enhancersequence directs expression of the marker in proopiomelanocortin neuronsin the arcuate nucleus, nucleus of the solitary tract, and immaturegranular layer neurons of the dentate gyrus of the hippocampus of themouse. A section of the arcuate nucleus of the mouse can be used todetermine if an agent affects caloric intake, food intake, appetite, orenergy expenditure.

A method is disclosed herein for screening for an agent that affectscaloric intake, appetite, energy expenditure or food intake. The methodincludes contacting a histological section of an arcuate nucleus from anon-human transgenic animal, with an agent to be tested.Proopiomelanocortin neurons in the histological section express aheterologous marker that distinguishes the proopiomelanocortin neuronsfrom other cells in the histological section. An electrophysiologicalresponse of a proopiomelanocortin neuron in the histological section isassessed, thereby determining if the agent affects caloric intake,appetite, energy expenditure, or food intake.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a set of diagrams and digital images showing the generation oftransgenic mice expressing EGFP in ARC, brainstem, and hippocampalneurons. FIG. 1 a is a schematic diagram of the structure of thePOMC-EGFP transgene. FIG. 1 b is a digital image showing theidentification of a single POMC neuron (arrowhead on recording electrodetip) by EGFP fluorescence (upper) and IR-DIC microscopy (lower) in aliving ARC slice prior to electrophysiological recordings. FIG. 1 c is aset of digital images showing the co-localization (bright, on right) ofEGFP (left) and β-endorphin immunoreactivity (middle) in ARC POMCneurons. Scale bars: b & c, 50 μm. FIG. 1 d is a set of diagrams showingthe distribution of EGFP-positive neuronal soma throughout the ARCnucleus. ◯=5 cells, ●=10 cells. FIG. 1 e is a digital image showing theidentification of POMC neurons by EGFP fluorescence in the medialnucleus of the solitary tract (SolM) adjacent to the central canal (CC)in the medullary region of the brainstem. FIG. 1 f is a lowermagnification digital image showing the identification of POMC neuronsby immunohistochemistry with an antisera directed against GFP in theSolM and area postrema (AP). FIGS. 1 g and 1 h are a set of digitalimages showing higher magnifications of the neurons in the SolM and AP,respectively, depicted in FIG. 1 f. FIGS. 1 i and 1 j are a set ofdigital images showing total nuclear staining with the fluorescent dyeHoechst 33258 and immature granular layer neurons by EGFP fluorescence,respectively, in the dentate gyrus (DG) of the hippocampus. FIG. 1 k isa digital image showing a higher magnification of the EGFP-positiveneurons in the DG depicted in FIG. 1 j. FIGS. 1 l and 1 m are a set ofdigital images showing the identification of immature granular layerneurons by immunohistochemistry with an antisera directed against GFP inthe DG of a 2 month old and an 18 month old transgenic mouse,respectively. FIG. 1 n is a digital image showing a higher magnificationof the EGFP-immunopositive neurons in the DG depicted in FIG. 1 l.

FIG. 2 is a tracing and graphs showing activation of MOP-Rshyperpolarizes the EGFP-labeled POMC neurons by opening Gprotein-coupled inwardly-rectifying potassium channels. FIG. 2 a is atracing showing met-enkephalin hyperpolarizes POMC neurons and inhibitsall action potentials. The horizontal bar indicates the time when 30 μMMet-Enk was bath-applied to the slice. FIG. 2 b is a graph showingmet-enkephalin current and reversal potential is shifted byextracellular K⁺ concentration. FIG. 2 c is a graph showingmet-enkephalin activates MOP-Rs on POMC neurons. A Met-Enk (30 μM)current was observed and the MOP-R specific antagonist CTAP (1 μM) wasapplied for 1 minute. Following CTAP Met-Enk elicited no current. Thefigure is representative of three experiments.

FIG. 3 are tracings and graphs demonstrating that leptin depolarizesPOMC neurons via a non-specific cation channel, and decreases GABAergictone onto POMC cells. FIG. 3 a is a tracing demonstrating that leptindepolarizes POMC neurons and increases the frequency of actionpotentials within 1 to 10 minutes of addition. The figure is arepresentative example of recordings made from 77 POMC neurons. FIG. 3 bis a graph showing that leptin causes a concentration dependentdepolarization of POMC cells. The depolarization caused by leptin wasdetermined at 0.1, 1, 10, 50, and 100 nM (EC₅₀=5.9 nM) in (8, 7, 9, 3,45) cells respectively. FIG. 3 c is a graph showing that leptindepolarizes POMC cells by activating a nonspecific cation current. Thefigure is representative of the response in 10 cells. FIG. 3 d is agraph showing that leptin decreases the frequency of IPSCs in POMCcells. The figure is an example of 5 cells in which leptin (100 nM)decreased the frequency of IPSCs. FIG. 3 e is a tracing demonstratingthat leptin had no effect on 5 adjacent non-fluorescent ARC neurons.FIG. 3 f is a tracing showing that leptin hyperpolarized 5non-fluorescent ARC neurons.

FIG. 4 is a set of images showing that the GABAergic inputs to POMCcells are from NPY neurons that co-express GABA. FIG. 4 a is a graphshowing that NPY decreases the frequency of mini IPSCs in POMC neurons.FIG. 4 b is a graph demonstrating that D-Trp⁸-γMSH (7 nM), a dose thatselectively activates MC3-R, increases the frequency of GABAergic IPSCsin POMC neurons. FIG. 4 c is a tracing showing that D-Trp⁸-γMSHhyperpolarizes POMC neurons. FIGS. 4 a, 4 b and 4 c are representative.FIG. 4 d is a set of digital images demonstrating that expression of NPYin nerve terminals adjacent to POMC neurons in the ARC. NPY nerveterminals (black, arrowheads); POMC neuronal soma (grey). Scale bar, 10μm. FIG. 4 e is a digital image showing expression of GABA and NPY innerve terminals synapsing onto POMC neurons in the ARC. GABAimmunoreactivity (10 nm gold particles, arrowheads without tail) and NPYimmunoreactivity (25 nm gold particles, arrows with tail) are inseparate vesicle populations co-localized within synaptic boutons thatmake direct contact with the soma of POMC neurons (DAB contrasted withuranyl acetate and lead citrate, diffuse black in cytoplasm). Scale bar,1 μm. FIG. 4 f is a diagram of the model of NPY/GABA and POMC neurons inthe ARC.

FIG. 5 is a set of digital images of c-fos expression in Pomc-EGFP mice.FIGS. 5 a and 5 b are digital images of representative sections (bregma−1.4 mm²²) of c-fos expression in the arcuate nucleus of Pomc-EGFP miceresponse to intraperitoneal saline (FIG. 5 a) or PYY₃₋₃₆ (5 μg/100 g)(FIG. 5 b). Scale bar 100 μm. 3V, third ventricle; Arc, arcuate nucleus.FIGS. 5 c and 5 d are digital images of representative sections showingPOMC-EGFP neurons (FIG. 5 c) and c-fos immunoreactivity (FIG. 5 d)either co-localising (bright arrows) or alone (single darker arrow).Scale bar 25 μm.

FIG. 6 is a set of images relating to the electrophysiological andneuropeptide responses to PYY₃₋₃₆ and Y2A. FIG. 6 a is a tracing showingthe effect of PYY₃₋₃₆ (10 nM) on the frequency of action potentials inPOMC neurons (whole-cell configuration recordings; n=22)*p<0.05. PYY₃₋₃₈was administered at time D for 3 minutes; baseline, −3 to 0 minute;PYY₃₋₃₆, 2-5 minutes; and wash-out, 8-11 minutes. Inset shows arepresentative recording of membrane potential and action potentialfrequency. FIG. 6 b is a graph of the effect if PYY₃₋₃₈ (10 nM) on thefrequency of action potentials in loose cell-attached patch recordings(n=8). Data from individual cells were normalized to the firing rate forthe 200 s before PYY₃₋₃₈ addition. FIG. 6 c is a tracing and a graph ofthe effect of PYY₃₋₃₈ (50 nM) on spontaneous IPSCs onto POMC neurons(n=13). Inset shows a representative recording of IPSCs before and afterPYY₃₋₃₆ (50 nM), respectively. Results in FIGS. 6 a-6 c are expressed asmean±s.e.m.

FIG. 7 is a schematic diagram of transgenes carrying variable lengths ordeletions of 5′ flanking sequences of the mouse POMC gene. The EGFP genewas inserted into the second exon immediately before the site oftranslational initiation. A polyadenylation signal from the large Tantigen of the SV40 virus was included immediately adjacent and 3′ tothe EGFP gene. Black boxes are mouse POMC exons. Open boxes, EGFP orLacZ as indicated. Striped boxes are nPOMC1, nPOMC2, and nPOMC3 sites.The white box is the TK minimal promoter in front of the hGH structuralgene. Restriction endonuclease sites used for subcloning include a BamHIsite at position approximately −9 kb and two Sma1 sites at positionsapproximately −6.5 kb and −0.7 kb upstream of the transcriptionalinitiation start site. Right: Plus signs indicate that the transgene isexpressed correctly in POMC pituitary corticotroph and melanotroph cells(PIT), POMC hypothalamic arcuate and nucleus tractus solitarius neurons(Arc/NTS), immature granular layer neurons of the dentate gyrus of thehippocampus (DG), or additional sites in the central nervous system(Add) and minus signs indicate the absence of expression. The one-stepPOMC expression cassette has a polylinker inserted into a StuIrestriction site located immediately 5′ to the translational start codonin exon 2.

FIG. 8 is a set of digital images of sections of the arcuate nucleus.FIG. 8 a is a digital image showing fluorescence in POMC neurons of thearcuate nucleus of the hypothalamus in a −13/+8 POMC-EGFP (delta−6.5/0.7) transgenic mouse. FIG. 8 b is a digital image showingimmunohistochemical localization of human growth hormone (hGH) using anantisera specific for hGH. POMC neurons in the arcuate nucleus of a−13/−9 POMC-TKhGH transgenic mouse express the hGH marker. 3V, thirdventricle.

FIG. 9 is a set of diagrams showing sequence alignments. FIG. 9 a is aPIP Maker multiple sequence alignment between 24 kb containing the humanPOMC gene, 4 kb of the mouse 5′ flanking region located between 9 and 13kb from the TATA box, and the three exons with short flanking sequencesobtained from GenBank (J00610, J00611, and J00612). Conserved regionsare indicated with horizontal black lines on gray shaded background.Exons 1, 2, and 3 are indicated; repetitive intergenic regions arepresent at −5 kb and −6 kb; two highly conserved intergenic regionslonger than 100 bp are identified as nPOMC1 and nPOMC2. The gray andwhite horizontal boxes indicated GC-rich regions. FIG. 9 b is a similaranalysis performed with the Dotter program using the 4 kb between −13and −9 of the mouse POMC gene and 15 kb of the human 5′ flanking region.Diagonal lines inside the gray-shaded areas indicate the conserved sitesnPOMC1 and nPOMC2.

FIG. 10 is the sequence alignments of nPOMC1 (FIG. 10 a) (5′ half, human(SEQ ID NO: 1), cow (SEQ ID NO: 2), hamster (SEQ ID NO: 3), mouse (SEDID NO: 4), and rat (SEQ ID NO: 5)), nPOMC1 (FIG. 10 b) (3′ half, human(SEQ ID NO: 6), mouse (SEQ ID NO: 7), and rat (SEQ ID NO: 8)), nPOMC1complete template (SEQ ID NO: 9) and nPOMC2 (FIG. 10 c) (human (SEQ IDNO: 10), cow (SEQ ID NO: 11), rabbit (SEQ ID NO: 12), mouse (SEQ ID NO:13), and rat (SEQ ID NO: 14)), nPOMC2 template (SEQ ID NO: 15) andnPOMC3 (FIG. 10 c) (human (SEQ ID NO: 16), mouse (SEQ ID NO: 17), andrat (SEQ ID NO: 18)), and nPOMC3 template (SEQ ID NO: 19). PPH2 and PPH3primers (underlined) were designed based on the sequences of human andmouse nPOMC1 (5′ half) and used to amplify the corresponding sequencesfrom cow and hamster genomic DNA. PPH8 and PPH9 primers (underlined)were designed based on the sequences of human and mouse nPOMC2 and usedto amplify the corresponding sequences from cow and rabbit genomic DNA.The consensus binding site for STAT3 (TTCCNGGAA) is shown adjacent toputative STAT3 binding sites within the nPOMC1 (3′ half) element. In thetemplates shown, in addition to the standard nucleotides (A, G, C, T)M=G/T, N=any nucleotide or a blank, P=A/T, W=A/G, X=C/G, Y=C/T, Z=A/C.

FIG. 11 is the nucleotide sequences of mouse (M. musculus), human (H.sapiens), and rat (R. norvegicus) nPOMC1 (FIG. 11 a), nPOMC2 (FIG. 11b), and nPOMC3 (FIG. 11 b) elements. NPOMC1 element from mousechromosome 12 nucleotides 3,808,013-3,808,447 (SEQ ID NO: 20), nPOMC1element from human chromosome 2 nucleotides 2,324,416-2,323,942 (SEQ IDNO: 21), and the nPOMC1 element from rat chromosome 6 nucleotides1,962,320-1,962,887 (SEQ ID NO: 22); nPOMC2 element from mousechromosome 12 nucleotides 3,810,489-3,810,724 (SEQ ID NO: 23), nPOMC2element from human chromosome 2 nucleotides 2,322,890-2,322,659 (SEQ IDNO: 24), and the nPOMC2 element from rat chromosome 6 nucleotides1,964,684-1,964,909 (SEQ ID NO: 25); nPOMC3 element from mousechromosome 12 nucleotides 3,813,451-3,813,596 (SEQ ID NO: 26), nPOMC3element from human chromosome 2 nucleotides 2,320,149-2,320,009 (SEQ IDNO: 27), and the nPOMC3 element from rat chromosome 6 nucleotides1,967,170-1,967,309 (SEQ ID NO: 28) are shown.

FIG. 12 is a set of graphs demonstrating that Ghrelin increases thesecretory activity of NPY neurons onto POMC neurons hyperpolarizes POMCneurons, and decreases the frequency of action potentials in POMCneurons. FIG. 12 a is a graph demonstrating that Ghrelin increases thefrequency of spontaneous synaptic GABA release onto POMC neurons.Results shown in the figure are representative of 18 experiments.Increased release of GABA from NPY neurons is shown, thus Ghrelin isincreasing the activity of NPY neurons. FIG. 12 b is a graphdemonstrating that Ghrelin mildly hyperpolarizes POMC neurons anddecreases the spontaneous activity of POMC neurons. Results shown in thefigure are representative of 34 experiments. FIG. 12 a and FIG. 12 bwere recorded in conventional whole cell mode. FIG. 12 c is a graphdemonstrating that Ghrelin decreases the frequency of action potentialsin POMC neurons, an effect that reverses with washout of the drug.Ghrelin induced a 50% decrease of the normalized mean (+/−s.e.m.) POMCneuron firing rate. These recordings were made in loose-cell-attachedmode.

FIG. 13 is a set of graphs demonstrating that fenfluramine (d-FEN)increases the frequency of action potentials and depolarizes POMCneurons. FIG. 13 a shows the results obtained using aloose-cell-attached mode. d-FEN (20 microM) induced a doubling of themean (+/−s.e.m.) POMC-neuron firing rate (n=3). This effect was reversedwith drug washout. FIG. 13 b is a graph of the mean (+/−s.e.m.) peakdepolarization of POMC neurons (n=4-8 per dose) bathed with d-FEN, 5-HTmCPP or MK 212 using conventional whole cell recordings.

FIG. 14 shows the sequences of the POMC regulatory region. FIG. 14 a isthe sequence of the mouse POMC regulatory region from −13 to −9kilobases (SEQ ID NO: 29). FIG. 14 b is the sequence of the human POMCpromoter region from −11.5 to −7 kilobases (SEQ ID NO: 30). FIG. 14 c isthe sequence of the rat POMC promoter region from −13.8 to −9.8kilobases (SEQ ID NO: 31). The nucleotide sequences of the nPOMC1 andnPOMC2 elements are highlighted in gray in FIGS. 14 a-14 c. FIGS. 14d-14 e are the sequence of the mouse POMC promoter region from −9 to−0.7 kilobases (SEQ ID NO: 32). FIGS. 14 f-14 g are the sequence of thehuman POMC promoter region from −7 to −0.7 kilobases (SEQ ID NO: 33).FIG. 14 f is the sequence of the rat POMC promoter region from −9.8 to−0.7 kilobases (SEQ ID NO: 34). The nucleotide sequences of the nPOMC3element is highlighted in gray in FIGS. 14 d-f.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand.

SEQ ID NO: 1 is the nucleic acid sequence of the 5′ half of the humannPOMC1.

SEQ ID NO: 2 is the nucleic acid sequence of the 5′ half of the cownPOMC1.

SEQ ID NO: 3 is the nucleic acid sequence of the 5′ half of the hamsternPOMC1.

SEQ ID NO: 4 is the nucleic acid sequence of the 5′ half of the mousenPOMC1.

SEQ ID NO: 5 is the nucleic acid sequence of the 5′ half of the rat POMCenhancer.

SEQ ID NO: 6 is the nucleic acid sequence of the 3′ half of the humannPOMC1.

SEQ ID NO: 7 is the nucleic acid sequence of the 3′ half of the mousenPOMC1.

SEQ ID NO: 8 is the nucleic acid sequence of the 3′ half of the ratnPOMC1.

SEQ ID NO: 9 is the nPOMC1 complete template based on multiple species.

SEQ ID NO: 10 is the nucleic acid sequence of the human nPOMC2.

SEQ ID NO: 11 is the nucleic acid sequence of the cow nPOMC2.

SEQ ID NO: 12 is the nucleic acid sequence of the rabbit nPOMC2.

SEQ ID NO: 13 is the nucleic acid sequence of the mouse nPOMC2.

SEQ ID NO: 14 is the nucleic acid sequence of the rat nPOMC2.

SEQ ID NO: 15 is the nPOMC2 complete template based on multiple species.

SEQ ID NO: 16 is the nucleic acid sequence of the human nPOMC3.

SEQ ID NO: 17 is the nucleic acid sequence of the mouse nPOMC3.

SEQ ID NO: 18 is the nucleic acid sequence of the rat nPOMC3.

SEQ ID NO: 19 is the nPOMC3 complete template based on multiple species.

SEQ ID NO: 20 is the nucleic acid sequence of the complete mouse nPOMC1.

SEQ ID NO: 21 is the nucleic acid sequence of the complete human nPOMC1.

SEQ ID NO: 22 is the nucleic acid sequence of the complete rat nPOMC1.

SEQ ID NO: 23 is the nucleic acid sequence of the complete mouse nPOMC2.

SEQ ID NO: 24 is the nucleic acid sequence of the complete human nPOMC2.

SEQ ID NO: 25 is the nucleic acid sequence of the complete rat nPOMC2.

SEQ ID NO: 26 is the nucleic acid sequence of the complete mouse nPOMC3.

SEQ ID NO: 27 is the nucleic acid sequence of the complete human nPOMC3.

SEQ ID NO: 28 is the nucleic acid sequence of the complete rat nPOMC3.

SEQ ID NO: 29 is the nucleic acid sequence of the mouse POMC 5′ flankingregion from approximately −13 to −9 kilobases from the transcriptionalstart site.

SEQ ID NO: 30 is the nucleic acid sequence of the human POMC 5′ flankingregion from approximately −11.5 to −7 kilobases from the transcriptionalstart site.

SEQ ID NO: 31 is the nucleic acid sequence of the rat POMC 5′ flankingregion from approximately −13.8 to −9.8 kilobases from thetranscriptional start site.

SEQ ID NO: 32 is the nucleic acid sequence of the mouse POMC 5′ flankingregion from approximately −9 to −0.7 kilobases from the transcriptionalstart site.

SEQ ID NO: 33 is the nucleic acid sequence of the human POMC 5′ flankingregion from approximately −7 to −0.7 kilobases from the transcriptionalstart site.

SEQ ID NO: 34 is the nucleic acid sequence of the rat POMC 5′ flankingregion from approximately −9.8 to −0.7 kilobases from thetranscriptional start site.

SEQ ID NO:35 is the consensus core binding site for STAT3.

SEQ ID NO:36 is a highly conserved palindromic oligonucleotide sequencewithin the nPOMC1 template (SEQ ID NO: 9).

SEQ ID NO: 37 is the sequence of an exemplary polylinker.

DETAILED DESCRIPTION I. Abbreviations

-   -   α-MSH: alpha melanocortin stimulating hormone    -   Arc: arcuate nucleus of the hypothalamus    -   CPP: m-CPP hydrochloride, 1-(3-Chlorophenyl)piperazine        5-HT_(2B/2C) receptor agonist    -   d-FEN: fenfluarmine    -   DG: dentate gyrus of the hippocampuis    -   EPSP: excitatory postsynaptic potential    -   GABA: γ-aminobutyric acid    -   GFP, EGFP: green fluorescent protein, enhanced green fluorescent        protein    -   hGH: human growth hormone    -   IPSC: inhibitory postsynaptic current    -   kb: kilobase    -   kg: kilogram    -   MOP-R: μ-opioid receptor    -   MK: MK212 hydrochloride, or 6-Chloro-2-(1-piperazinyl)pyrazine        5-HT_(2C) serotonin receptor agonist.    -   MV: millivolts    -   nPOMC1: neural POMC regulatory element 1    -   nPOMC2: neural POMC regulatory element 2    -   nPOMC3: neural POMC regulatory element 3    -   NPY: neuropeptide Y    -   NTS: nucleus tractus solitarius    -   pmol: picomole    -   POMC: proopiomelanocortin    -   RIA: radioimmunoassay    -   RPA: RNase protection assay    -   s.e.m.: standard error of the mean    -   TH: tyrosine hydroxylase    -   μM: micromolar    -   V: volts    -   Y2A: N-acetyl (Leu²⁸, Leu³¹) NPY (24-36)

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Action potential: A rapidly propagated electrical message that speedsalong an axon of a neuron and over the surface membrane of many muscleand glandular cells. In axons they are brief, travel at constantvelocity, and maintain a constant amplitude. Like all electricalmessages of the central nervous system, the action potential is amembrane potential change caused by the flow of ions through ionchannels in the membrane. In one embodiment, an action potential is aregenerative wave of sodium permeability.

Affinity Tag: A nucleic acid sequence which can be included in a vectorwhich can aid in the purification of a protein encoded by the vector.The term affinity tag refers to the nucleic acid sequence for the tag,and the tag protein sequence encoded by the nucleic acid sequence.Examples of affinity tags include, but are not limited to: histidine,such as 6× histidine, S-tag, glutathione-S-transferase (GST) andstreptavidin.

Agent: Any polypeptide, compound, small molecule, organic compound,salt, polynucleotide, or other molecule of interest.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibiotic Resistance Cassette: A nucleic acid sequence encoding aselectable marker which confers resistance to that antibiotic in a hostcell in which the nucleic acid is translated. Examples of antibioticresistance cassettes include, but are not limited to: kanamycin,ampicillin, tetracycline, chloramphenicol, neomycin, hygromycin, andzeocin.

Antagonist: A substance that tends to nullify the action of another, asan agent that binds to a cell receptor without eliciting a biologicalresponse, blocking binding of substances that could elicit suchresponses.

Appetite: A natural desire, or longing for food. In one embodiment,appetite is measured by a survey to assess the desire for food.Increased appetite generally leads to increased feeding behavior.

Binding: A specific interaction between two molecules, such that the twomolecules interact. Binding can be specific and selective, so that onemolecule is bound preferentially when compared to another molecule. Inone embodiment, specific binding is identified by a disassociationconstant (K_(d)).

An oligonucleotide binds or stably binds to a target nucleic acid if asufficient amount of the oligonucleotide forms base pairs or ishybridized to its target nucleic acid, to permit detection of thatbinding. Binding can be detected by physical or functional properties ofthe target: oligonucleotide complex. Binding between a target and anoligonucleotide can be detected by any method known to one skilled inthe art, including functional and physical binding assays. Binding canbe detected functionally by determining whether binding has anobservable effect upon a biosynthetic process such as expression of agene, DNA replication, transcription and translation.

Physical methods of detecting the binding of complementary strands ofDNA or RNA are well known in the art, and include such methods as DNaseI or chemical foot printing, gel shift and affinity cleavage assays,Northern blotting, dot blotting and light absorption detectionprocedures. For example, a method which is widely used, because it issimple and reliable, involves observing a change in light absorption ofa solution containing an oligonucleotide (or an analog) and a targetnucleic acid at 220 to 300 nm as the temperature is slowly increased. Ifthe oligonucleotide or analog has bound to its target, there is a suddenincrease in absorption at a characteristic temperature as theoligonucleotide (or analog) and target dissociate or melt.

The binding between an oligomer and its target nucleic acid isfrequently characterized by the temperature (T_(m)) at which 50% of theoligomer is melted from its target. A higher T_(m) means a stronger ormore stable complex relative to a complex with a lower T_(m).

CAAT box: An upstream promoter element generally located at −75 to −80relative to the RNA start site. It influences the frequency ofinitiation, most likely by acting directly on the basal transcriptionfactors to enhance their assembly into an initiation complex. Thesequences between the CAAT and TATA elements are irrelevant and thedistance between them is flexible. The separation between the CAAT andTATA elements can usually be changed by 10 to 30 base pairs beforerendering them inoperable.

Caloric intake or calorie intake: The number of calories (energy)consumed by an individual.

Calorie: A unit of measurement in energy. A standard calorie is definedas 4.184 absolute joules, or the amount of energy it takes to raise thetemperature of one gram of water from 15° C. to 16° C. (or 1/100th theamount of energy needed to raise the temperature of one gram of water atone atmosphere pressure from 0° C. to 100° C.). Food calories areactually equal to 1,000 standard calories (1 food calorie=1kilocalorie).

Conserved Enhancer Substitution: A modification made in an enhancersequence that does not alter the ability of the sequence to directexpression of an operably linked nucleic acid sequence. Modificationsinclude substitutions (base replacements), insertions, and/or deletionsof nucleic acid residues. In several specific, non-limiting examples,conserved enhancer substitutions include at most about fifty, such as atmost about one, at most about two, at most about five, at most aboutten, at most about fifteen, or at most about twenty base substitutionsin a POMC enhancer element. A POMC enhancer element includes, but is notlimited to, an element with a sequence set forth as SEQ ID NO: 9, SEQ IDNO: 15, or SEQ ID NO: 19. In several other specific non-limitingexamples conserved enhancer substitutions include at most about fiftysubstitutions in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.In other specific, non-limiting examples, conserved enhancersubstitutions include at most fifty, such as one, at most two, at mostfive, at most ten, or at most twenty insertions or deletions in any oneof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. Additional examples aredescribed below.

Conservative variation: The replacement of an amino acid residue byanother, biologically similar residue. Examples of conservativevariations include the substitution of one hydrophobic residue such asisoleucine, valine, leucine or methionine for another, or thesubstitution of one polar residue for another, such as the substitutionof arginine for lysine, glutamic for aspartic acid, or glutamine forasparagine, and the like. The term “conservative variation” alsoincludes the use of a substituted amino acid in place of anunsubstituted parent amino acid provided that antibodies raised to thesubstituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

Non-limiting examples of conservative amino acid substitutions includethose listed below:

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Current: The amount of charge per unit time. Current is generated in acell membrane of a neuron by an action potential or by opening of ionchannels in the cell membrane and serves to depolarize or hyperpolarizeadjacent membrane areas.

Deletion: The removal of a sequence of nucleic acid, such as DNA, theregions on either side being joined together.

DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid, RNA). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine, and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides,referred to as codons, in DNA molecules code for amino acid in apolypeptide. The term codon is also used for the corresponding (andcomplementary) sequences of three nucleotides in the mRNA into which theDNA sequence is transcribed.

Depolarization: An increase in the membrane potential of a cell. Certainstimuli reduce the charge across the plasma membrane. These can beelectrical stimuli (which open voltage-gated channels), mechanicalstimuli (which activate mechanically-gated channels) or certainneurotransmitters (which open ligand-gated channels). In each case, thefacilitated diffusion of sodium into the cell increases the restingpotential at that spot on the cell creating an excitatory postsynapticpotential (EPSP). Depolarizations can also be generated by decreasingthe frequency of inhibitory postsynaptic currents (IPSCs), these are dueto inhibitory neurotransmitters facilitating the influx of chloride ionsinto the cell, creating an IPSC. If the potential is increased to thethreshold voltage (about −50 mV in mammalian neurons), an actionpotential is usually generated in the cell.

Electroporation: A method of inducing or allowing a cell to take upmacromolecules by applying electric fields to reversibly permeabilizethe cell walls. Various methods and apparatuses used are further definedand described in: U.S. Pat. No. 4,695,547; U.S. Pat. No. 4,764,473; U.S.Pat. No. 4,946,793; U.S. Pat. No. 4,906,576; U.S. Pat. No. 4,923,814;and U.S. Pat. No. 4,849,089.

Enhancer: A cis-regulatory sequence that can elevate levels oftranscription of a coding sequence from an adjacent promoter. Manytissue specific enhancers can determine spatial patterns of geneexpression in higher eukaryotes. Enhancers can act on promoters overmany tens of kilobases of DNA and can be 5′ or 3′ to the promoter theyregulate. Enhancers can function either by initiating transcription froma promoter operably linked to the enhancer or by providing binding sitesfor gene regulatory proteins that increase transcription of a minimalpromoter.

Eukaryotic cell: A cell having an organized nucleus bounded by a nuclearmembrane. These include simpler organisms such as yeasts, slime molds,and the like, as well as cells from multicellular organisms such asinvertebrates, vertebrates, and mammals. Multicellular organisms includea variety of cell types, such as: endothelial cell, smooth muscle cell,epithelial cell, hepatocyte, cells of neural crest origin, tumor cell,hematopoetic cell, immunologic cell, T cell, B cell, monocyte,macrophage, dendritic cell, fibroblast, keratinocyte, neuronal cell,glial cell, adipocyte, myoblast, myocyte, chondroblast, chondrocyte,osteoblast, osteocyte, osteoclast, secretory cell, endocrine cell,oocyte, and spermatocyte. These cell types are described in standardhistology texts, such as McCormack, Introduction to Histology, (c) 1984by J.P. Lippincott Co.; Wheater et al., eds., Functional Histology, 2ndEd., (c) 1987 by Churchill Livingstone; Fawcett et al., eds., Bloom andFawcett: A Textbook of Histology, (c) 1984 by William and Wilkins.

Exon: A portion of a gene whose nucleotide sequence is transcribed byRNA polymerase and is present in both the primary heteronuclear RNAtranscript and the mature messenger RNA following splicing and deletionof the transcribed intron sequences. An exon (or portion of an exon) canbe either nontranslated and contain regulatory information forprocessing or stabilization of the RNA or translated by ribosomalcomplexes into an encoded protein sequence.

Food intake: The amount of food consumed by an individual. Food intakecan be measured by volume or by weight. In one embodiment, food intakeis the total amount of food consumed by an individual. In anotherembodiment, food intake is the amount of proteins, fat, carbohydrates,cholesterol, vitamins, minerals, or any other food component, of theindividual. “Protein intake” refers to the amount of protein consumed byan individual. Similarly, “fat intake,” “carbohydrate intake,”“cholesterol intake,” “vitamin intake,” and “mineral intake” refer tothe amount of proteins, fat, carbohydrates, cholesterol, vitamins, orminerals consumed by an individual.

Gene: A DNA sequence that comprises control and coding sequencesnecessary for the production of a polypeptide or protein. Thepolypeptide can be encoded by a full-length coding sequence or by anyportion of the coding sequence in some embodiments, so long as at leasta portion of the desired activity of the polypeptide is retained. A“foreign gene” is any nucleic acid (e.g., gene sequence) that isintroduced into the genome of an animal by experimental manipulationsand can include gene sequences found in that animal so long as theintroduced gene contains some modification (e.g., a point mutation, thepresence of a selectable marker gene, a non-native regulatory sequence,or a native sequence integrated into the genome at a non-nativelocation, etc.) relative to the naturally-occurring gene.

Hyperpolarization: A decrease in the membrane potential of a cell.Inhibitory neurotransmitters inhibit the transmission of nerve impulsesvia hyperpolarization. This hyperpolarization is called an inhibitorypostsynaptic potential (IPSP). Although the threshold voltage of thecell is unchanged, a hyperpolarized cell requires a stronger excitatorystimulus to reach threshold.

Inhibitory PostSynaptic Current: A current that inhibits anelectrophysiological parameter of a postsynaptic cell. The potential ofa postsynaptic cell can be analyzed to determine an effect on apresynaptic cell. In one embodiment, the postsynaptic cell is held involtage clamp mode, and postsynaptic currents are recorded. Ifnecessary, antagonists of other classes of current can be added. In onespecific, non-limiting example, to record GABAergic IPSCs, blockers ofexcitatory channels or receptors can be added. The instantaneousfrequency over time is then determined.

In one embodiment, IPSCs give a measure of the frequency of GABA releasefrom an NPY neuron. Thus, as NPY neurons release GABA onto POMC neurons,measurement of IPSC frequency is a gauge of the inhibitory tone thatPOMC neurons are receiving, and can be used to assess the effect of anagent that affects an NPY neuron, such as an antagonist or agonist ofPYY.

Intron: An intragenic nucleic acid sequence in eukaryotes that is notexpressed in a mature RNA molecule. Introns of the present disclosureinclude full-length intron sequences, or a portion thereof, such as apart of a full-length intron sequence.

In vitro amplification: Techniques that increases the number of copiesof a nucleic acid molecule in a sample or specimen. An example ofamplification is the polymerase chain reaction, in which a biologicalsample collected from a subject is contacted with a pair ofoligonucleotide primers, under conditions that allow for thehybridization of the primers to nucleic acid template in the sample. Theprimers are extended under suitable conditions, dissociated from thetemplate, and then re-annealed, extended, and dissociated to amplify thenumber of copies of the nucleic acid. The product of in vitroamplification may be characterized by electrophoresis, restrictionendonuclease cleavage patterns, oligonucleotide hybridization orligation, and/or nucleic acid sequencing, using standard techniques.Other examples of in vitro amplification techniques include stranddisplacement amplification (see U.S. Pat. No. 5,744,311);transcription-free isothermal amplification (see U.S. Pat. No.6,033,881); repair chain reaction amplification (see WO 90/01069);ligase chain reaction amplification (see EP-A-320 308); gap fillingligase chain reaction amplification (see U.S. Pat. No. 5,427,930);coupled ligase detection and PCR (see U.S. Pat. No. 6,027,889); andNASBA™ RNA transcription-free amplification (see U.S. Pat. No.6,025,134).

Isolated: An isolated biological component (such as a nucleic acid,peptide or protein) has been substantially separated, produced apartfrom, or purified away from other biological components in the cell ofthe organism in which the component naturally occurs, i.e. otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins that have been isolated include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids, peptides, and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids. The term further includes a nucleic acid sequenceisolated from the adjacent 5′ and/or 3′ nucleic acid sequences as foundin the endogenous chromosomal location.

Marker: A protein, or a gene encoding a protein, for which a system isavailable to identify cells that produce the protein. Specificnon-limiting examples of a marker include drug resistance markers, suchas G148 or hygromycin. Additionally, a marker can be a protein or a geneencoding a protein for which negative selection can be used to identifythe cell expressing the marker. A specific, non-limiting examples of anegative selection marker includes, but is not limited to, the HSV-tkgene. This gene will make the cells sensitive to agents such asacyclovir and gancyclovir. Another specific, non-limiting example of aselectable marker is a protein, or a gene encoding a protein, whereinselection can be made by using a cell surface marker, for example, toselect overexpression of the marker by fluorescence activated cellsorting (FACS). In another specific, non-limiting example of aselectable marker is a protein, or a gene encoding a protein, that canbe identified in a cell based on its fluorescent or enzymaticproperties. Specific, non-limiting examples include, but are not limitedto, enhanced fluorescent green protein (EGFP), alkaline phosphatase, orhorseradish peroxidase. A marker can also be a polypeptide or antigenicepitope thereof, wherein an antibody that specifically binds thepolypeptide can be used to identify cells that express the polypeptideor antigenic epitope. One specific, non-limiting example of apolypeptide of use is human growth Hormone (hGH).

Membrane potential: The electrical potential of the interior of the cellwith respect to the environment, such as an external bath solution. Oneof skill in the art can readily assess the membrane potential of a cell,such as by using conventional whole cell techniques. Activation of acell is associated with less negative membrane potentials (for exampleshifts from about −50 mV to about −40 mV). These changes in potentialincrease the likelihood of action potentials, and thus lead to anincrease in the rate of action potentials.

The rate of action potentials can be assessed using many approaches,such as using conventional whole cell access, or using, for example,perforated-patch whole-cell and cell-attached configurations. In eachevent the absolute voltage or current is not assessed, rather thefrequency of rapid deflections characteristic of action potentials isassessed, as a function of time (therefore this frequency is aninstantaneous frequency, reported in “bins”). This time component can berelated to the time at which a compound, such as a PYY agonist, isapplied to the bath to analyze the effect of the compound, such as thePYY agonist, on action potential firing rate.

Neuropeptide Y (NPY): A 36-amino acid peptide that is a neuropeptideidentified in the mammalian brain. NPY is believed to be an importantregulator in both the central and peripheral nervous systems andinfluences a diverse range of physiological parameters, includingeffects on psychomotor activity, food intake, central endocrinesecretion, and vasoactivity in the cardiovascular system. Highconcentrations of NPY are found in the sympathetic nerves supplying thecoronary, cerebral, and renal vasculature and have contributed tovasoconstriction. NPY binding sites have been identified in a variety oftissues, including spleen, intestinal membranes, brain, aortic smoothmuscle, kidney, testis, and placenta. In addition, binding sites havebeen reported in a number of rat and human cell lines.

NPY binds to several receptors, including the Y1, Y2, Y3, Y4 (PP), Y5,Y6, and Y7 receptors. These receptors are recognized based on bindingaffinities, pharmacology, and sequence (if known). Most, if not all ofthese receptors are G protein coupled receptors. One of skill in the artcan readily determine the dissociation constant (K_(d)) value of a givencompound for a Y receptor. This value is dependent on the selectivity ofthe compound tested. For example, a compound with a K_(d) which is lessthan 10 nM is generally considered an excellent drug candidate. However,a compound that has a lower affinity, but is selective for theparticular receptor, can also be a good drug candidate. In one specific,non-limiting example, an assay, such as a competition assays, is used todetermine if a compound of interest is a Y2 receptor agonist. Assaysuseful for evaluating neuropeptide Y receptor antagonists are also wellknown in the art (see U.S. Pat. No. 5,284,839, which is hereinincorporated by reference, and Walker et al., Journal of Neurosciences8:2438-2446, 1988).

Oligonucleotide: A linear polynucleotide sequence of up to about 200nucleotide bases in length, for example a polynucleotide (such as DNA orRNA) which is at least 6 nucleotides, for example at least 15, 50, 100or even 200 nucleotides long.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

Polylinker: An artificial nucleic acid sequence of about twenty tothirty nucleotides in length that is designed to include restrictionsites cleaved by several restriction enzymes. A specific non-limitingexample of a polylinker is: 5′-GCCCGGGCTCGAGTTTAAAGCGCGC-3′ (SEQ ID NO:36), that includes the restrictions sites for SrfI, SmaI, XhoI, DraI,and, BssHII.

Polypeptide: A polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred. The terms “polypeptide” or“protein” as used herein are intended to encompass any amino acidsequence and include modified sequences such as glycoproteins. The term“polypeptide” is specifically intended to cover naturally occurringproteins, as well as those which are recombinantly or syntheticallyproduced. The term “polypeptide fragment” refers to a portion of apolypeptide, for example such a fragment which exhibits at least oneuseful sequence in binding a receptor. The term “functional fragments ofa polypeptide” refers to all fragments of a polypeptide that retain anactivity of the polypeptide. Biologically functional peptides can alsoinclude fusion proteins, in which the peptide of interest has been fusedto another peptide that does not decrease its desired activity.

Promoter: An array of nucleic acid control sequences which directtranscription of a nucleic acid. In one embodiment, a promoter includesnecessary nucleic acid sequences near the start site of transcription,such as, in the case of a polymerase II type promoter, a TATA element.Enhancer and repressor elements can be located adjacent to, or distal tothe sequences necessary for the start site of transcription, and can belocated as much as several thousand base pairs from the start site oftranscription. A “heterologous promoter” is a promoter from one geneoperably linked to a control element or a protein coding sequence fromanother gene or another species of animal. In one specific, non-limitingexample, a POMC enhancer is operably linked to a heterologous promoter(e.g. a promoter that is not from a POMC gene from the same species ofanimal), such as an SV40, thymidine kinase, beta actin, tyrosinehydroxylase, or other promoter. In another specific non-limitingexample, a heterologous promoter is not a POMC promoter.

A promoter can be a “strong” promoter, which promotes transcription ofRNA at high levels, for example at levels such that the transcriptionalactivity of the promoter generally accounts for about 25% oftranscriptional activity of all transcription within a cell. Thestrength of a promoter is often tissue-specific and thus can vary fromone cell type to another. For example, the promoter of the humancytomegalovirus early gene 1 is a classic strong promoter because itgenerates high levels of transcriptional activity in many cell types.

In other embodiments, the promoter is a “tissue-specific promoter,”which promotes transcription in a single cell type or narrow range oftissues. In one embodiment, a tissue specific promoter promotesexpression in the pituitary and/or the hypothalamus, but not in othertissues. In another embodiment, a tissue specific promoter promotesexpression in the pituitary, but not in other tissues. In a furtherembodiment, a tissue specific promoter promotes expression only in alimited subset of neurons (e.g. in the arcuate nucleus of thehypothalamus and nucleus tractus solitarius), but not in other tissues(e.g., heart, lung, pancreas, intestines, skin, etc.)

In other embodiments, the promoter is a “minimal” promoter, which hasvery low intrinsic transcriptional activity in the absence of operablylinked enhancer sequences. A minimal promoter is one that does not haveinherent cell-specific or tissue-specific activity, but may directtranscriptional initiation in multiple eukaryotic cell types whenoperably linked to a cell- or tissue-specific enhancer sequence. Onespecific, non-limiting example of a minimal promoter is the minimalpromoter sequences of the herpes simplex virus type 1thymidine kinase(HSV1-tk) gene.

A “POMC promoter” is the array of nucleic acid control sequences thatdirect transcription of a POMC nucleic acid in the endogenouschromosomal location of a species of interest. In one embodiment, a POMCpromoter includes necessary nucleic acid sequences near the start siteof transcription, such as a polymerase II type promoter, a TATA element,and a SP1 site. In one specific, non-limiting example, in the rat, thePOMC promoter is located at the 5′ end of the rat POMC gene. In onespecific, non-limiting example the rat POMC promoter is theapproximately 700, or approximately 400, base pairs immediately 5′ ofthe transcription start site (Hammer et al., Molec. Endocrinol. 4:1689,1990), such as the sequence located from −323 to −34. The rat POMCpromoter cooperatively directs transcription to corticotrophs andmelanotrophs in transgenic mice. In the rat, the activator SP1 interactswith a GC-rich region in the promoter (see Liu et al., Biochem. J.312:827-832, 1995).

Proopiomelanocortin (POMC): A glycosylated protein of a molecular weightof 31 kDa. POMC has been demonstrated to be synthesized mainly in theanterior pituitary, in the hypothalamus, and in the brainstem. However,other tissues, cell types, or neurons also express the POMC gene, albeitat lower levels. This protein is a precursor protein, post-translationalprocessing of POMC yields several neuroactive peptides upon specificcleavage. The POMC coding sequence includes the amino acid sequences ofadrenocorticotropic (ACTH) hormone and beta-lipotropin. ACTH isprocessed to produce the polypeptides melanotropin (alpha-MSH) andcorticotrophin-like intermediate lobe peptide (CLIP). Beta-lipotropin isprocessed to produce the peptides γ-lipotropin, beta-endorphins, andbeta-melanocyte stimulating hormone (β-MSH). The amino-terminal fragmentof POMC is processed to a family of gamma-MSH peptides and to a peptidewith putative mitogenic stimulatory activity of the adrenal corticalcells. The biological activity of POMC-derived peptides is furtherregulated in a tissue-specific manner by acetylation of theamino-terminal amino acid residue and/or amidation of thecarboxyterminal amino acid residue by the enzymepeptidyl-α-monooxygenase (PAM).

The POMC gene (human chromosome 2p23) contains three exons and twointrons: one, of about 3.5 kb, interrupts the N-terminal fragment of thecommon precursor mostly encoded in exon 3. Exon 2 contains the sequencefor a portion of the 5′ untranslated portion of the mRNA, all of thesignal sequence which directs insertion of the precursor protein intothe endoplasmic reticulum, and 8 amino acids of the N-terminal fragment.The overall arrangement of introns and exons in the POMC gene is almostidentical in all mammalian species. Hormonal control of POMC genetranscription and release of peptide products derived from the POMCprecursor is tissue-specific; for example, glucocorticoids specificallyinhibit anterior but not intermediate pituitary POMC transcription.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the protein is more pure than the protein inits natural environment within a cell. Such proteins may be produced,for example, by standard purification techniques, or by recombinantexpression. In some embodiments, a preparation of a protein is purifiedsuch that the protein represents at least 50%, for example at least 70%,of the total protein content of the preparation.

PYY: A peptide YY polypeptide obtained or derived from any species.Thus, PYY includes the human full length polypeptide (as set forth inSEQ ID NO: 1) and species variations of PYY, including e.g. murine,hamster, chicken, bovine, rat, and dog PYY. In one embodiment, PYYagonists do not include NPY. A “PYY agonist” is any compound which bindsto a receptor that specifically binds PYY, and elicits an effect of PYY.In one embodiment, a PYY agonist is a compound that affects food intake,caloric intake, or appetite, and/or which binds specifically in a Yreceptor assay or competes for binding with PYY, such as in acompetitive binding assay with labeled PYY. PYY agonists include, butare not limited to, compounds that bind to the Y2 receptor.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g. by genetic engineering techniques, such as thosedescribed in Sambrook et al. (in Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, N.Y., 1989).

Restriction Enzyme: An enzyme that cleaves a specific nucleic acidsequence. A restriction enzyme usually recognizes a specific shortsequence of bases in the DNA. Different restriction enzymes recognizedifferent sequences, i.e., they have different specificities.Consequently, restriction enzymes allow DNA to be cleaved at specific,pre-determined locations. The sequence which a restriction enzymerecognizes and digests the nucleic acid is called a restriction site.The process of cutting the DNA is called a restriction digest or adigestion. Specific, non-limiting examples of restriction sites are EcoRI or Bgl II, or a StuI site, where the first part of the name refers tothe strain of bacteria which was the source of the enzyme (e.g.,Escherichia coli RY 13) and the second part of the name is a Romannumeral.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the percentage of conservation between thesequences, otherwise referred to as sequence identity. Sequence identityis frequently measured in terms of percentage identity (or similarity orhomology); the higher the percentage, the more similar the two sequencesare. Homologues or variants of a POMC sequence will possess a relativelyhigh degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet., 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet. Other specific, non-limiting examplesof sequence alignment programs specifically designed to identifyconserved regions of genomic DNA of greater than or equal to 100nucleotides are PIPMaker (Schwartz et al., Genome Research 10: 577-586,2000) and DOTTER (Erik et al., Gene 167: GC1-10, 1995).

Homologues and variants of a POMC enhancer sequence are typicallycharacterized by possession of at least 75%, for example at least 80%,90%, 95%, 98%, or 99%, sequence identity counted over the full lengthalignment with the originating POMC enhancer sequence using the NCBIBlast 2.0, set to default parameters. Methods for determining sequenceidentity over such short windows are available at the NCBI website onthe internet. One of skill in the art will appreciate that thesesequence identity ranges are provided for guidance only; it is entirelypossible that strongly significant homologues could be obtained thatfall outside of the ranges provided.

Substantially purified: A polypeptide which is substantially free ofother proteins, lipids, carbohydrates or other materials with which itis naturally associated. For example, the polypeptide may be at least50%, 80% or 90% free of other proteins, lipids, carbohydrates or othermaterials with which it is naturally associated.

TATA box: An upstream promoter element; a DNA conserved sequence TATAAAthat is generally located −25 to −30 base pairs relative to thetranscriptional start site. This sequence is part of the promotersequence of eukaryotic genes and binds transcription factor IID (TFIID).RNA polymerase recognizes the TFIID-TATA protein-DNA complex. The TATAbox sequence is critical both for promoter activity and for determiningthe exact point of RNA chain initiation. For example, in the human POMCgene the sequence of the TATA box is TATATAA and is located 28 bases 5′to the transcriptional start site. In the mouse POMC gene the TATA boxsequence is TATAAAA and is located 30 bases 5′ of the transcriptionalstart site.

Transcriptional enhancement: A property of producing an increase in therate of transcription of linked sequences that contain a functionalpromoter.

Transcriptional unit: A polynucleotide sequence that includes the entirecoding region, including all exons and introns, the translational startand stop codons, and the cleavage and polyadenylation consensussequence.

Unless specified otherwise, the left-hand end of single-strandedpolynucleotide sequences is the 5′ end; the left-hand direction ofdouble-stranded polynucleotide sequences is referred to as the 5′direction. The direction of 5′ to 3′ addition of nascent RNA transcriptsis referred to as the transcription direction; sequence regions on theDNA strand having the same sequence as the RNA and which are 5′ of the5′ end of the RNA transcript are referred to as “upstream sequences”;sequence regions on the DNA strand having the same sequence as the RNAand which are 3′ to the 3′ end of the RNA transcript are referred to as“downstream sequences”.

Transduced and Transfected: A virus or viral vector transduces a cellwhen it transfers nucleic acid into the cell. A cell is “stablytransduced” by a nucleic acid transduced into the cell when the DNAbecomes stably replicated by the cell, either by incorporation of thenucleic acid into the cellular genome, or by episomal replication. Asused herein, the terms transduced and transfected encompass alltechniques by which a nucleic acid molecule might be introduced intosuch a cell, including transfection with viral vectors, transfectionwith plasmid vectors, and introduction of naked DNA by electroporation,lipofection, injection, and particle gun acceleration.

Transgene: A foreign gene that is placed into an organism by introducingthe foreign gene into embryonic stem (ES) cells, newly fertilized eggsor early embryos. In one embodiment, a transgene is a gene sequence, forexample a sequence that encodes a marker polypeptide that can bedetected using methods known to one of skill in the art. In anotherembodiment, the transgene encodes a therapeutic polypeptide that can beused to alleviate or relieve a symptom of a disorder. In yet anotherembodiment, the transgene encodes a therapeutically effectiveoligonucleotide, for example an antisense oligonucleotide, whereinexpression of the oligonucleotide inhibits expression of a targetnucleic acid sequence. In a further embodiment, the transgene encodes anantisense nucleic acid or a ribozyme. In yet another embodiment, atransgene is a stop cassette.

In other embodiments, a transgene contains regulatory sequences (e.g. anenhancer, such as a POMC enhancer) operably linked to a transcriptionalunit. Thus, the transgene can include regulatory sequences operablylinked to a nucleic acid sequence encoding a polypeptide, such as amarker.

Transgenic Cell: Cells that contain foreign, non-native DNA.

Transgenic Animal: An animal, for example, a non-human animal such as amouse, that has had DNA introduced into one or more of its cellsartificially. By way of example, this is commonly done by randomintegration or by targeted insertion. DNA can be integrated in a randomfashion by injecting it into the pronucleus of a fertilized ovum. Inthis case, the DNA can integrate anywhere in the genome, and multiplecopies often integrate in a head-to-tail fashion. There is no need forhomology between the injected DNA and the host genome. In most cases,the foreign transgene is transmitted to subsequence generations in aMendelian fashion (a germ-line transgenic).

Targeted insertion, the other common method of producing transgenicanimals, is accomplished by introducing the DNA into embryonic stem (ES)cells and selecting for cells in which the DNA has undergone homologousrecombination with matching genomic sequences. For this to occur, thereoften are several kilobases of homology between the exogenous andgenomic DNA, and positive selectable markers are often included. Inaddition, negative selectable markers are often used to select againstcells that have incorporated DNA by non-homologous recombination (randominsertion).

Vector: A nucleic acid molecule used to introduce foreign DNA into acell, thereby producing a transfected cell. A vector can include nucleicacid sequences that permit it to replicate in the cell, such as anorigin of replication. A vector can also include one or more marker ortherapeutic transgenes and other genetic elements known in the art.

In some embodiments, the vector is a non-viral vector, such as abacterial plasmid vector. In other embodiments, the vector is a viralvector. Examples of viral vectors include, but are not limited toadenoviral vectors, retroviral vectors, and Herpes viral vectors.

Voltage: An electric potential or potential difference, expressed involts.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

POMC Enhancer Elements

POMC neurons of the hypothalamus are critical components of the neuralcircuitry controlling appetite, feeding, and metabolism. In addition,POMC-derived peptides synthesized in neurons of the central nervoussystem participate in the control of body temperature, blood pressure,stress-induced analgesia, reproductive function, cognitive abilities,emotional states, rewarding behavior, responses to drugs of abuse,including opioids and ethanol, and neuroimmunomodulation. They alsomodulate the activity of other hypothalamic neurons which controlreproduction, thyroid hormone levels, growth hormone secretion,prolactin secretion, and neuroendocrine stress responses. These neuronsalso are involved in the nervous system's endogenous analgesic andreward circuits.

Disclosed herein are the sequences of several mammalian POMC enhancerelements. These sequences are of use in directing expression of a geneproduct to the POMC neurons. Generally, a POMC enhancer includes atleast one NPOMC1, nPOMC2, or an nPOMC3 element. A POMC enhancer can alsoinclude at least two nPOMC1, nPOMC2, or nPOMC3 elements. A POMC enhancercan also include an nPOMC1, nPOMC2, and an nPOMC3 element. Templates fornPOMC1 (SEQ ID NO: 9), nPOMC2 (SEQ ID NO: 15) and nPOMC3 (SEQ ID NO: 19)are shown in FIGS. 10 a-10 c. The sequences of several exemplary nPOMC1,nPOMC2, and nPOMC3 elements are also shown in FIGS. 10 a-c. Transgenesincluding a POMC enhancer element are diagrammed in FIG. 7.

In one specific, non-limiting example, the enhancer is approximately 4kilobases in length that is located upstream of the POMC codingsequences of the endogenous chromosomal of the mammal of interest. Inother specific, non-limiting examples, the enhancer includes an nPOMC1element and an nPOMC2 element, an nPOMC1 element and an nPOMC3 element,an nPOMC2 and an nPOMC3 element, or an nPOMC1, nPOMC2, and an nPOMC3element. Generally, any combination of these elements is of use,provided they direct expression to POMC neurons. In yet anotherspecific, non-limiting example, a POMC enhancer does not include a POMCpromoter. Generally, a POMC promoter includes necessary nucleic acidsequences near the start site of transcription, such as a polymerase IItype promoter, a TATA element, and a SP1 site near the POMC start oftranscription (as measured from the endogenous chromosomal location). Inone specific, non-limiting example, in the rat, the POMC promoter islocated at the 5′ end of the rat POMC protein coding sequences. In onespecific, non-limiting example the rat POMC promoter is theapproximately 700, or approximately 400, base pairs immediately 5′ ofthe transcription start site (Hammer et al., Molec. Endocrinol. 4:1689,1990), such as the sequence located from −323 to −34. The rat POMCpromoter cooperatively directs transcription to corticotrophs andmelanotrophs in transgenic mice. Similarly, sequences from the mousePOMC gene containing 2 kb of 5′ flanking region (Rubinstein et al.,Neuroendocrinology 58:373-380; 1993) or from the human POMC genepromoter containing 2.9 kb of the 5′ flanking region of the human gene(Tsukada et al., DNA Cell Biol. 13:755-762; 1994) are active inpituitary cells.

A template for the complete nPOMC1 sequence based on sequencecomparisons among multiple mammalian species is set forth as SEQ ID NO:9. In one embodiment, an nPOMC1 sequence is at least 90% identical toSEQ ID NO: 9, such as a sequence 95% identical, 98% identical, or 99%identical to SEQ ID NO: 9. In another embodiment, an nPOMC1 sequenceincludes at most fifty conserved enhancer substitutions of SEQ ID NO: 9,such as, but not limited to, at most about two, at most about five, atmost about ten, at most about twenty, or at most about fifty conservedenhancer substitutions of SEQ ID NO: 9.

Several specific nPOMC1 elements are disclosed herein. These sequencesinclude the 5′ half of the nPOMC element, which includes SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. Thesesequences can also include the 3′ half of an nPOMC1 element, as setforth as SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. In oneembodiment, an nPOMC1 sequence also includes the 3′ half of the element,as set forth as SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. In severalembodiments, an nPOMC1 sequence includes SEQ ID NO: 1 and SEQ ID NO: 6,SEQ ID NO: 4 and SEQ ID NO: 7, or SEQ ID NO: 5 and SEQ ID NO: 8. Inother embodiments, the nPOMC1 element includes any one of SEQ ID NOs:1-5 in combination with any one of SEQ ID NOs: 6-8.

It should be noted that modifications can be made in these sequencesthat do not alter their ability to direct expression of a heterologousgene in POMC neurons. Modifications include substitutions, insertions,and/or deletions of nucleic acid residues. Although these POMC sequencesdo not encode proteins, and thus do not fit under the classicaldefinition of conservative substitutions (see above), thesesubstitutions can be considered “conserved enhancer” substitutions. Inseveral specific, non-limiting examples, conserved enhancersubstitutions include at most fifty, such as at most about one, at mostabout two, at most about five, at most about ten, or at most abouttwenty substitutions in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ IDNO: 8. In other specific, non-limiting examples, conserved enhancersubstitutions include at most one, at most two, at most five, at mostten, or at most twenty deletions in any one of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7or SEQ ID NO: 8.

As shown in FIGS. 10 a-10 b, several conserved areas of nPOMC1 have beenidentified (see black and gray boxes). Thus, in several specific,non-limiting examples, conserved enhancer substitutions include at mostone, at most two, at most five, at most ten, or at most twentysubstitutions outside of conserved areas any one of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7 or SEQ ID NO: 8. For example, in SEQ ID NO: 1, the first nucleicacid reside (a guanine, G), can be replaced by a cytosine (C), adenine(A), or thymine (T). Similarly, in SEQ ID NO: 1, the first nucleic acidresidue can be deleted, yet the sequence can still be used toappropriately direct expression in a POMC neuron. Similarly, the thirdnucleic acid residue, “A,” can be replaced by a G, C, or T, as long asthe nucleic acid sequence can be used to appropriately direct expressionto a POMC neuron. In another example, nucleic acid residue 21 in SEQ IDNO: 2, “T” can be replaced by an A, G, C, or deleted. In addition, othernucleic acid elements can be added to these sequences withoutinterfering with their function, such as promoter sequences.

In another embodiment, an nPOMC1 sequence of use includes an element atleast about 70% homologous with the corresponding originating POMCsequence. For example, a sequence at least 80%, at least 90%, at least95%, at least 98%, or at least 99% homologous with the correspondingoriginating POMC sequence, such as SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, orSEQ ID NO: 8. In one specific, non-limiting example, identical regions(shown in black, FIGS. 10 a-b) are retained. In other, specific,non-limiting examples identical regions between two or more (shown ingray are retained). In a further, non-limiting example, non-conservedregions (shown white) are substituted to create the sequences with thedesired identity to the originating sequences.

A template for the complete nPOMC2 sequence based on sequencecomparisons among multiple mammalian species is set forth as SEQ ID NO:15. In one embodiment, an nPOMC2 sequence is about 90% identical to SEQID NO: 17, such as about 95% identical, 98% identical, or 99% identicalto SEQ ID NO: 15. In another embodiment, an nPOMC2 sequence includes atmost twenty conserved enhancer substitutions of SEQ ID NO: 15, such asat most two, at most five, at most ten, or at most fifteen conservedenhancer substitutions of SEQ ID NO: 15.

Several specific nPOMC2 elements are disclosed herein. These sequencesinclude, but are not limited to, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 13, and SEQ ID NO: 14. It should be noted thatmodifications can be made in these nPOMC2 sequences that do not altertheir ability to direct expression of a heterologous gene in POMCneurons. Modifications include substitutions, insertions, and/ordeletions of nucleic acid residues. In several specific, non-limitingexamples, conserved enhancer substitutions include at most twentyconserved enhancer substitutions, such as at most one, at most two, atmost five, at most ten, or at most fifteen substitutions in any one ofSEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ IDNO: 14. In other specific, non-limiting examples, conserved enhancersubstitutions include at most one, at most two, at most five, at mostten, or at most twenty deletions in any one of SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.

As shown in FIG. 10 c, several conserved areas of nPOMC2 have also beenidentified (see black and gray boxes). Thus, in several specific,non-limiting examples, conserved enhancer substitutions include at mostone, at most two, at most five, at most ten, or at most twentysubstitutions outside of conserved areas any one of SEQ ID NO: 10, SEQID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14. For example,in SEQ ID NO: 10, the fourth nucleic acid residue (a thymine, T) can bereplaced by a cytosine (C), adenine (A), or guanine (G). Similarly, inSEQ ID NO: 11, the eleventh nucleic acid residue can be deleted, yet thesequence can still be used to appropriately direct expression in a POMCneuron. Similarly, the eleventh nucleic acid residue in SEQ ID NO: 11,“G,” can be replaced by an A, C, or T, as long as the nucleic acidsequence can be used to appropriately direct expression to a POMCneuron. In another example, nucleic acid residue 9 in SEQ ID NO: 13, “T”can be replaced by an A, G, C or deleted. In addition, other nucleicacid elements can be added to these sequences without interfering withtheir function, such as, but not limited to, promoter sequences.

In another embodiment, an nPOMC2 sequence of use includes an element atleast 70% homologous with the corresponding originating nPOMC2 sequence.For example, a sequence at least 80%, at least 90%, at least 95%, atleast 98%, or at least 99% homologous with the corresponding originatingnPOMC2 sequence, such as SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, and SEQ ID NO: 14. In one specific, non-limiting example,identical regions (shown in black, FIG. 10 c) are retained. In other,specific, non-limiting examples identical regions between two or more(shown in gray are retained). In a further, non-limiting example,non-conserved regions (shown white) are substituted to create thesequences with the desired identity to the originating sequences.

A template for the complete nPOMC3 sequence based on sequencecomparisons among multiple mammalian species is set forth as SEQ ID NO:9. In several examples, an nPOMC3 sequence is at least about 90%identical to SEQ ID NO: 19. In other examples, an nPOMC sequence isabout 95% identical, 98% identical, or 99% identical to SEQ ID NO: 19.In another embodiment, an nPOMC3 sequence includes at most fifteenconserved enhancer substitutions of SEQ ID NO: 19, such as at most 2, atmost 5, at most ten, at most twelve conserved enhancer substitutions ofSEQ ID NO: 19.

Several specific nPOMC3 elements are disclosed herein. These sequencesinclude, but are not limited to, SEQ ID NO: 16, SEQ ID NO: 17, and SEQID NO: 18. It should be noted that modifications can be made in thesenPOMC3 sequences that do not alter their ability to direct expression ofa heterologous gene in POMC neurons. As described above, modificationsinclude substitutions, insertions, and/or deletions of nucleic acidresidues. In several specific, non-limiting examples, conserved enhancersubstitutions include at most about twenty substitutions, such as atmost one, at most two, at most five, at most ten, or at most fifteensubstitutions in SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. Inother specific, non-limiting examples, conserved enhancer substitutionsinclude at most one, at most two, at most five, at most ten, or at mosttwenty deletions in any one of SEQ ID NO: 16, SEQ ID NO: 17, or SEQ IDNO: 18.

As shown in FIG. 10 c, several conserved areas of nPOMC3 have also beenidentified (see black or gray boxes). Thus, in several specific,non-limiting examples, conserved enhancer substitutions include at mostone, at most two, at most five, at most ten, or at most twentysubstitutions outside of conserved areas of SEQ ID NO: 16, SEQ ID NO:17, or SEQ ID NO: 18. For example, in SEQ ID NO: 16, the first nucleicacid residue a “C,” can be replaced by a G, T, or an A, and/or thesecond nucleic acid residue, a “T,” can be replaced by a G, A, or a C,and/or the third nucleic acid residue, a “G,” can be replaced by an A,T, or C. Alternatively, the first, second, and/or third nucleic acidresidues can be deleted. Similarly, in SEQ ID NO: 17, the first nucleicacid reside a “C,” can be replaced by a G, T, or an A, and/or the secondnucleic acid residue, a “T,” can be replaced by an A, G, or a C, and/orthe third nucleic acid residue, a “G,” can be replaced by an A, T, or C,as long as the sequence can still be used to appropriately directexpression in a POMC neuron. In addition, other nucleic acid elementscan be added to these sequences without interfering with their function,such as, but not limited to, promoter sequences.

In another embodiment, an nPOMC3 sequence of use includes an element atleast 70% homologous with the corresponding originating POMC sequence.For example, a sequence at least 80%, at least 90%, at least 95%, atleast 98%, or at least 99% homologous with the corresponding originatingPOMC sequence, such as SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.In one specific, non-limiting example, identical regions (shown inblack, FIG. 10 c) are retained. In other, specific, non-limitingexamples identical regions between two or more (shown in gray areretained). In a further, non-limiting example, non-conserved regions(shown white) are substituted to create the sequences with the desiredidentity to the originating sequences.

In several other embodiments, an isolated POMC enhancer includes severalkilobases (kb) of sequence 5′ (upstream) of the POMC promoter and thetranscription start site (which is generally considered to start atnucleotide 1 of the coding sequence) of the POMC gene. Thus, in onespecific, non-limiting example, a POMC enhancer includes the nucleotidesequences from about −13 kilobases relative to the transcription startsite and the transcription start site. This genomic fragment of about 13kb directs eutopic expression of operably linked genes to neurons of thearcuate nucleus of the hypothalamus and the nucleus of the tractussolitarius and to pituitary corticotrophs and melanotrophs, all celltypes where the endogenous POMC gene is normally expressed.

In another specific, non-limiting example, a POMC enhancer includes thenucleotide sequences from about −13 kilobases to about −0.7 kilobasesrelative to the transcription start site of the mouse POMC gene orequivalent mammalian gene sequence (e.g. mouse, rat, cow, hamster orhuman sequence). In a further specific, non-limiting example, a POMCenhancer includes the nucleotide sequence located between about −9kilobases to about −0.7 kilobases relative to the transcription startsite of a mouse proopiomelanocortin gene, or any other mammalian species(e.g. rat, cow, hamster, human, etc.). In a further specific,non-limiting example, a POMC enhancer includes the nucleotide sequencelocated from about −13 kilobases to about −6.5 kilobases of an upstreamregion of a start site of a proopiomelanocortin protein coding sequence.In yet another non-limiting example, a POMC enhancer includes thenucleotide sequence located from about −13 kilobases to about −9kilobases of an upstream region relative to the transcription start siteof a mouse proopiomelanocortin gene, or any other mammalian species(e.g. rat, cow, hamster, human, etc.). Each of these enhancers directeutopic expression of operably linked genes to neurons of the arcuatenucleus of the hypothalamus and the nucleus of the tractus slitarius,and/or to pituitary corticotrophs and melanotrophs. All of these celltypes express the endogenous POMC gene.

A human large genomic contig (Homo sapiens chromosome 2 referencecontig) including the complete POMC gene and the nPOMC1, nPOMC2 andnPOMC3 elements can be found in GenBank, the NIH sequence databasemaintained by the National Center for Biotechnology Information (NCBI).The information relating to this sequence is as follows: Accession No.:NT_(—)005204.

A mouse large genomic contig (Mus musculus chromosome 12 WGSsupercontig) including the complete POMC gene and the nPOMC1, POMC2 andnPOMC3 elements) can also be found in GenBank. The information relatingto this sequence is as follows: Accession No.: NW_(—)000041.

A rat large genomic contig (Rattus norvegicus chromosome 6 WGSsupercontig) including the complete POMC gene and the nPOMC1, POMC2 andnPOMC3 elements) can also be found in GenBank. The information relatingto this sequence is as follows: Accession No.: NW_(—)043940.

All these GenBank entries (human, mouse, and rat) are incorporatedherein by reference in their entirety. Using the information providedherein, and the information provided in GenBank, one of skill in the artcan readily isolate the relevant sequences.

The POMC enhancer sequences described above can be obtained by manymethods. The more common include chemical synthesis by known methodssuch as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as described in EP 266,032 published May 4,1988, or deoxynucleoside H-phosphonate intermediates as described byFroehler et at., Nucl. Acids Res. 14:5399-5407, 1986. A POMC enhancersequences can also be amplified directly from the genomic DNA using thepolymerase chain reaction (PCR) as described in U.S. Pat. No. 4,683,195issued Jul. 28, 1987. Finally, the desired nucleotide sequence (whetherdouble or single stranded) can be readily synthesized by any number ofcommercial suppliers such as Genset (San Diego, Calif.) or Clontech(Palo Alto, Calif.). Commercial suppliers, as well as anyonesynthesizing or PCR amplifying the sequence themselves, can create thesequences with particular requested overhangs to match any particularcloning needs.

Expression Systems

A POMC enhancer can be included in an expression vector to directexpression of a heterologous nucleic acid sequence. Thus otherexpression control sequences including appropriate promoters,transcription terminators, a start codon (i.e., ATG) in front of aprotein-encoding gene, splicing signal for introns, maintenance of thecorrect reading frame of that gene to permit proper translation of mRNA,and stop codons can be included with a POMC enhancer in an expressionvector. Generally expression control sequences include a promoter, aminimal sequence sufficient to direct transcription.

The expression vector typically contains an origin of replication, apromoter, as well as specific genes which allow phenotypic selection ofthe transformed cells. Vectors suitable for use include, but are notlimited to the pMSXND expression vector for expression in mammaliancells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988). Generally, theexpression vector will include a promoter. The promoter can be inducibleor constitutive. The promoter can be tissue specific. Suitable promoterinclude the thymidine kinase promoter (TK), metallothionein I,polyhedron, neuron specific enolase, tyrosine hydroxylase, beta-actin,or other promoters. In one embodiment the promoter is a POMC promoter.In another embodiment, the promoter is a heterologous promoter.

In one example, the POMC enhancer is located upstream of the desiredpromoter, but enhancer elements can generally be located anywhere on thevector and still have an enhancing effect. However, the amount ofincreased activity will generally diminish with distance. Additionally,two or more copies of a POMC enhancer sequence can be operably linkedone after the other to produce an even greater increase in promoteractivity.

Generally, an expression vector includes a nucleic acid sequenceencoding a polypeptide of interest. A polypeptide of interest can be apolypeptide that affects a function of the transfected cell.Polypeptides of interest include, but are not limited to, polypeptidesthat confer antibiotic resistance, receptors, oncogenes, andneurotransmitters. A polypeptide of interest can also be a markerpolypeptide, which is used to identify a cell of interest. Markerpolypeptides include fluorescent polypeptides, enzymes, or antigens thatcan be identified using conventional molecular biology procedures. Forexample, the polypeptide can be a fluorescent marker (e.g., greenfluorescent protein, Aequoria Victoria, or Discosoma DSRed), anantigenic markers (e.g., human growth hormone, human insulin, human HLAantigens), a cell surface marker (e.g., CD4, or a any cell surfacereceptor), or an enzymatic marker (e.g., lacZ, alkaline phosphatase).Techniques for identifying these markers in host cells includeimmunohistochemistry and fluorescent microscopy, and are well known inthe art.

RNA molecules transcribed from an expression vector need not always betranslated into a polypeptide to express a functional activity. Specificnon-limiting examples of other molecules of interest include antisenseRNA molecules complementary to an RNA of interest, ribozymes, smallinhibitory RNAs, and naturally occurring or modified tRNAs.

Expression vectors including a POMC enhancer can be used to transformhost cells. Hosts can include isolated microbial, yeast, insect andmammalian cells, as well as cells located in the organism. Biologicallyfunctional viral and plasmid DNA vectors capable of expression andreplication in a host are known in the art, and can be used to transfectany cell of interest. Where the cell is a mammalian cell, the geneticchange is generally achieved by introduction of the DNA into the genomeof the cell (i.e., stable) or as an episome.

A “transfected cell” is a cell into which (or into an ancestor of which)has been introduced, by means of recombinant DNA techniques, a DNAmolecule including a POMC enhancer element. Transfection of a host cellwith recombinant DNA may be carried out by conventional techniques asare well known to those skilled in the art. Where the host isprokaryotic, such as E. coli, competent cells which are capable of DNAuptake can be prepared from cells harvested after exponential growthphase and subsequently treated by the CaCl₂ method using procedures wellknown in the art. Alternatively, MgCl₂ or RbCl can be used.Transformation can also be performed after forming a protoplast of thehost cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences including the POMC enhancer, and asecond foreign DNA molecule encoding a selectable phenotype, such asneomycin resistance. Another method is to use a eukaryotic viral vector,such as simian virus 40 (SV40) or bovine papilloma virus, to transientlyinfect or transform eukaryotic cells and express the protein (see forexample, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory,Gluzman ed., 1982). Other specific, non-limiting examples of viralvectors include adenoviral vectors, lentiviral vectors, retroviralvectors, and pseudorabies vectors.

The POMC enhancer elements disclosed herein can also be used in theproduction of transgenic animals such as transgenic mice, as describedbelow.

Transgenic Animals

In one embodiment, in order to direct expression of a marker into POMCneurons, a non-human animal is generated that carries a transgenecomprising a nucleic acid encoding a polypeptide operably linked to aPOMC nucleic acid sequence, including a POMC enhancer element. In onespecific, non-limiting example, expression of the marker distinguishesthe POMC neurons from all other cells within the arcuate nucleus.

The POMC enhancer elements disclosed herein can be used to directexpression of a marker in POMC neurons in the arcuate nucleus of thenon-human animal. The POMC enhancer element is operably linked to apromoter. Specific promoters of use include, but are not limited to, atissue specific promoter such as the POMC promoter. Specific promotersof use also include a constitutive promoter, such as, but not limited tothe thymidine kinase promoter or the human ββ-globin minimal, or anactin promoter, amongst others.

The promoter is operably linked to a nucleic acid encoding a marker,such as a polypeptide. The marker can be, but is not limited to, anypolypeptide of interest. Markers include, but are not limited to,fluorescent markers (e.g., green fluorescent protein, Aequoria Victoria,or Discosoma DSRed), antigenic markers (e.g., human growth hormone,human insulin, human HLA antigens), cell surface markers (e.g., CD4, ora any cell surface receptor), or enzymatic markers (e.g., lacZ). Inanother non-limiting example the promoter is operably linked to induceexpression of a marker that is a functionally active RNA molecule (e.g.antisense RNA molecules, ribozymes, small inhibitory RNAs, and naturallyoccurring or modified tRNAs).

The cDNA that encodes the marker can be fused in proper reading frameunder the transcriptional and translational control of regulatorysequence of interest, such as a promoter and a POMC enhancer sequencethat directs expression of the marker in the POMC neurons of the arcuatenucleus of the hypothalamus and the nucleus of the tractus solitarius.The sequences include, but are not limited to, an nPOMC1, nPOMC2, and/ornPOMC3 element(s), as disclosed herein, that direct expression of themarker in the POMC neurons of the arcuate nucleus. Specific,non-limiting examples of POMC sequences of use include, but are notlimited to, murine, human, bovine, hamster, and rabbit POMC sequences.Variants of these POMC sequences, such as, but not limited to deletions,insertions, and additions are also of use, provided that these variantsare conserved enhancer substitutions that direct expression of thepolypeptide of interest in POMC neurons of interest (see above). In oneembodiment, the POMC sequences can include the nPOMC1, nPOMC2 sequences,and/or the POMC promoter (see the Examples section below), provided thatthe sequences are included in a recombinant vector. In the chromosomallocation of the endogenous POMC gene, intervening sequences are foundbetween the POMC enhancer and the POMC promoter. For example, in themouse, the POMC enhancer elements are located between approximately −13kb and −6.5 kb upstream of the initiation site, while the POMC promoteris located in the region of −4.7 to 0 upstream of the initiation site.Thus, intervening sequences are located between position −6.5 to −0.7upstream of the initiation site. Thus, in one embodiment, an POMCenhancer element is operably linked to a POMC promoter, and at least aportion of the intervening sequences are not included in the construct.Thus, for some sequences (e.g. mouse), at least a portion, such as about1 kb, about 2 kb, about 3 kb, about 4 kb, about 5 kb, or about 6 kb ofintervening sequences are not included in the transgene.

This construct can be introduced into a vector to produce a product thatis then amplified, for example, by preparation in a bacterial vector,according to conventional methods (see, for example, Sambrook et al.,Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, 1989).The amplified construct is thereafter excised from the vector andpurified for use in producing transgenic animals.

Any animal can be of use in the methods disclosed herein, provided theanimal is any non-human animal. A “non-human animal” includes, but isnot limited to, a non-human primate, a farm animal such as swine,cattle, and poultry, a sport animal or pet such as dogs, cats, horses,hamsters, rodents, or a zoo animal such as lions, tigers, or bears. Inone specific, non-limiting example, the non-human animal is a transgenicanimal, such as, but not limited to, a transgenic mouse, cow, sheep, orgoat. In one specific, non-limiting example, the transgenic animal is amouse.

A transgenic animal contains cells that bear genetic informationreceived, directly or indirectly, by deliberate genetic manipulation atthe subcellular level, such as by microinjection or infection withrecombinant virus, such that a recombinant DNA is included in the cellsof the animal. This molecule can be integrated within the animal'schromosomes, or can be included as an extrachromosomally replicating DNAsequences, such as might be engineered into yeast artificialchromosomes. A transgenic animal can be a “germ cell line” transgenicanimal, such that the genetic information has been taken up andincorporated into a germ line cell, therefore conferring the ability totransfer the information to offspring. If such offspring in fact possesssome or all of that information, then they, too, are transgenic animals.

Transgenic animals can readily be produced by one of skill in the art.For example, transgenic animals can be produced by introducing intosingle cell embryos DNA encoding a marker, in a manner such that thepolynucleotides are stably integrated into the DNA of germ line cells ofthe mature animal and inherited in normal Mendelian fashion. Advances intechnologies for embryo micromanipulation permit introduction ofheterologous DNA into fertilized mammalian ova. For instance, totipotentor pluripotent stem cells can be transformed by microinjection, calciumphosphate mediated precipitation, liposome fusion, retroviral infectionor other means, the transformed cells are then introduced into theembryo, and the embryo then develops into a transgenic animal. In onenon-limiting method, developing embryos are infected with a retroviruscontaining the desired DNA, and transgenic animals produced from theinfected embryo.

In another, specific, non-limiting example, the appropriate DNA(s) areinjected into the pronucleus or cytoplasm of embryos, preferably at thesingle cell stage, and the embryos allowed to develop into maturetransgenic animals. These techniques are well known. For instance,reviews of standard laboratory procedures for microinjection ofheterologous DNAs into mammalian (mouse, pig, rabbit, sheep, goat, cow)fertilized ova include: Hogan et al., Manipulating the Mouse Embryo,Cold Spring Harbor Press, 1986; Krimpenfort et al., Bio/Technology 9:86,1991; Palmiter et al., Cell 41:343, 1985; Kraemer et al., GeneticManipulation of the Early Mammalian Embryo, Cold Spring HarborLaboratory Press, 1985; Hammer et al., Nature 315:680, 1985; Purcel etal., Science 244:1281, 1986; Wagner et al., U.S. Pat. No. 5,175,385;Krimpenfort et al., U.S. Pat. No. 5,175,384.

The disclosure of the neural-specific regulatory elements of the POMCgene is disclosed herein. This discovery was based on a combination oftwo approaches used in a sequential and iterative process (see theexamples section below). A functional screen of reporter gene expressionin transgenic mice was used to identify the broad regions of the POMCgene necessary for neural expression. A computational comparativegenomics approach was also applied to narrow the search for highlyconserved DNA sequences across species. The POMC sequences can be usedfor (1) development of transgenic animal models with heterologousprotein expression targeted specifically to POMC neurons; (2)development of genetic screening for human POMC alleles which carrypolymorphisms in the neural regulatory elements and affect the level ofPOMC gene expression and POMC neuropeptides in the central nervoussystem; (3) development of novel therapeutics based on small moleculesthat modulate POMC gene expression in the brain by interaction with theneural-specific DNA elements; and (4) identification of noveltranscription factors and intracellular signaling pathways and moleculesin POMC neurons that control the expression of the POMC gene orotherwise control the production, processing, or release of thebiologically active neuropeptide products from the neuronal terminals.

As disclosed herein, expression of a marker in a POMC neuron allowsspecific identification of these neurons in a histological section. Assuch, sections from a non-human animal expressing a marker inproopiomelanocortin (POMC) neuron can be used to identify agentsaffecting either POMC neurons directly or indirectly (e.g. agents actingon NPY neurons that make synaptic contact with nearby POMC neurons) asthe ability to identify the POMC neurons allow the measurement ofspecific neurophysiological parameters of these cells. The ability toidentify and measure electrophysiological parameters of the POMC neuronsenables screening for agents that affect these cells, such as agentsthat affect caloric intake, food intake, or appetite. In addition, theability of agents to alter the electrophysiological parameters of thePOMC neurons provides an assay to screen for agents that affect thecontrol of body temperature, blood pressure, stress-induced analgesia,reproductive function, cognitive abilities, emotional states, rewardingbehavior, responses to drugs of abuse, including opioids and ethanol,and neuroimmunomodulation

Methods for Screening

Methods for screening for an agent that affects caloric intake, foodintake, appetite, and/or energy expenditure are disclosed herein.Methods are also disclosed to screen for an agent that affects bodytemperature, blood pressure, stress-induced analgesia, reproductivefunction, cognitive abilities, emotional states, rewarding behavior,responses to drugs of abuse, including opioids and ethanol, andneuroimmunomodulation. The methods include contacting a histologicalsection of an arcuate nucleus from a non-human animal expressing amarker in proopiomelanocortin (POMC) neurons with the agent to betested. The expression of the marker distinguishes theproopiomelanocortin neurons from the other neurons (and other cells) inthe arcuate nucleus, such that electrophysiological measurements can bemade on the POMC neurons. An electrophysiological parameter of the POMCneurons is measured. The effect of the agent on this parameter indicatedif the agent has an effect on appetite, caloric intake, food intake, orenergy expenditure upon administration of a therapeutically effectiveamount of the agent to a subject. Similarly, an effect on anelectrophysiological parameter can indicate that an agent affects bodytemperature, blood pressure, stress-induced analgesia, reproductivefunction, cognitive abilities, emotional states, rewarding behavior,responses to drugs of abuse, including opioids and ethanol, andneuroimmunomodulation

A histological section of the arcuate nucleus from a non-human animalexpressing a marker in the POMC neurons is prepared using methods knownto one of skill in the art, and the section is contacted with a testagent of interest. The marker is essential for the identification of thePOMC neurons. An electrophysiological parameter of a POMC neuron is thenassessed. Suitable electrophysiological parameters include, but are notlimited to, hyperpolarization of the membrane potential of the POMCneuron and/or an increase in IPSCs in the POMC neuron. In onenon-limiting example, an agonist is selected that causeshyperpolarization of the membrane potential of a POMC neuron, andincreases IPSCs in a POMC neuron. Thus, an agent that affects appetite,caloric intake, food intake, energy expenditure, or can be selected.Similarly, and agent that affects body temperature, blood pressure,stress-induced analgesia, reproductive function, cognitive abilities,emotional states, rewarding behavior, responses to drugs of abuse,including opioids and ethanol, and neuroimmunomodulation can beselected.

One of skill in the art can readily assesses neuron firing rate,membrane voltage, depolarization, action potentials, and IPSC frequency.Exemplary methods are described in the examples section below. However,the methods disclosed herein are not limited to the devices andmeasurements described in the Examples section. For example, anyelectrophysiology amplifier can be utilized, such as, but not limitedto, devices produced by Dagan Instruments, Minneapolis, Minn., or HekaElektronik, Lambrecht/Pfalz, Germany.

In one embodiment, the membrane potential, action potential rate, and/orthe frequency of IPSCs in a POMC neuron treated with an agent iscompared to a control. Suitable controls include, but are not limitedto, a section contacted with a buffer alone, in the absence of an agent,a sample contact with a control agent, such as an agent known to have aneffect on the frequency of IPSCs, action potential rate, or to altermembrane potential of a POMC neuron, or an agent known not to have aneffect on IPSCs, action potential rate, or membrane potential of a POMCneuron.

In one specific, non-limiting example, a section of the arcuate nucleusis contacted with an agent, and the effect on the membrane potential ofa POMC neuron is measured. In this example, a change in the membranepotential of about 2 to about 50 mV indicates that the agent affects theactivity of POMC neurons and therefore affects food intake, caloricintake, appetite, and/or energy expenditure when administered to asubject. In another specific, non-limiting example, a change in IPSCfrequency is measured. In this example, a change in the IPSC frequencyis measured. In this example, a change of the IPSC frequency from about2% to a ten fold increase, or completely stopping IPSCs indicates thatthe agent affects food intake, caloric intake, appetite, and/or energyexpenditure. In another embodiment, a change in the action potentialrate of a POMC neuron is measured. In this example, a change in theaction potential rate of about 2% to completely stopping, or a change inthe action potential rate of greater than, or equal to, about a 20-fold,50-fold or 100-fold increase indicates that the agent affects foodintake, caloric intake, appetite, and/or energy expenditure.Alternatively, a change from no firing to activity of a POMC neuronindicates that the agent affects food intake, caloric intake, appetite,and/or energy expenditure. Other approaches to measuring activityinclude, but not be limited to, an analysis of the expression of c-fos.

One of skill in the art can readily identify a statistically analysis ofuse in assessing data obtained from the methods disclosed herein. Thestatistical analyses are standard, such as tests for repeatability, forexample analysis of variance, or wilcoxin signed rank test, areperformed, using an appropriate confidence level, such as, but notlimited to, p<0.05.

It should be noted that parameters of a POMC neuron, such as, but notlimited to, ion fluxes (e.g., a potassium flux), enzyme activation(e.g., a serine/threonine kinase), changes in cyclic nucleotides (e.g.,cAMP, cADP, cGMP, cGDP, etc.), among others, can also be measured. Aspecific, non-limiting example of a signaling event is the generation ofa K⁺ flux following the interaction of an agent with a POMC neuron. A“physiological indicator,” which is any compound in which a measurableproperty changes in a response to a physical parameter of the cell, canbe used to measure the signaling event. One specific, non-limitingexample of a measurable property is a change is in fluorescence of aphysiological indicator in response to an ion flux.

Fluorescence is one spectral property that can be used as the means ofdetecting a physiological parameter of a cell. A “fluorescent property”refers to the molar extinction coefficient at an appropriate excitationwavelength, the fluorescence quantum efficiency, the shape of theexcitation spectrum or emission spectrum, the excitation wavelengthmaximum and emission wavelength maximum, the ratio of excitationamplitudes at two different wavelengths, the ratio of emissionamplitudes at two different wavelengths, the excited state lifetime, orthe fluorescence anisotropy. A measurable difference in any one of theseproperties between a cell contacted with an agent as compared to acontrol cell suffices to identify a compound as being of interest. Ameasurable difference can be determined by determining the amount of anyquantitative fluorescent property, e.g., the amount of fluorescence at aparticular wavelength, or the integral of fluorescence over the emissionspectrum. Optimally, the physiological indicator is selected to havefluorescent properties that are easily distinguishable. A specific,non-limiting example of a fluorescent indicator of use is fura-2. Thisdye measures intracellular calcium. Increased intracellular calcium isan indicator of increased neuronal activity, while decreasedintracellular calcium is an indicator of decreased neural activity.

Any agent can be screened using the methods disclosed herein todetermine if it affects body temperature, blood pressure, stress-inducedanalgesia, reproductive function, cognitive abilities, emotional states,rewarding behavior, responses to drugs of abuse, including opioids andethanol, and neuroimmunomodulation.

In addition, an agent can be screened to determine if it affectsappetite, food intake, caloric intake, and/or energy metabolism.Suitable test agents include, but are not limited to, agents that bind,or are suspected of binding a receptor on either a POMC neuron, or a NPYneuron. Receptors on a POMC neuron include, but are not limited to amelanocortin receptor, a μ-opioid receptor, a leptin receptor, and aninsulin receptor. Receptors on a NPY neuron include, but are not limitedto, a Y2 receptor, a leptin receptor, an insulin receptor, amelanocortin receptor, or an opiod receptor. In one specific,non-limiting example the agent is a receptor agonist, or is suspected ofbeing a receptor agonist. In another specific, non-limiting example, theagent is a Y2 receptor agonist, or is suspected of being a Y2 receptoragonist.

Agents that can be tested using the methods disclosed herein includepolypeptides, chemical compounds; biological agents such as, but notlimited to polypeptides, cytokines, and small molecules,peptidomimetics; antibodies; and synthetic ligands, amongst others.Receptor agonists and antagonists can be screened.

“Incubating” includes conditions that allow contact between the testcompound and the histological section. “Contacting” includes in solutionand solid phase. The test compound may also be a combinatorial libraryfor screening a plurality of compounds. Compounds that are polypeptidesthat are identified in the method of the invention can be furtherevaluated, detected, cloned, sequenced, and the like, either in solutionof after binding to a solid support, by any method usually applied tothe detection of a specific DNA sequence, such as PCR, oligomerrestriction (Saiki et al., Bio/Technology 3:1008-1012, 1985),allele-specific oligonucleotide (ASO) probe analysis (Conner et al.,Proc. Natl. Acad. Sci. U.S.A. 80:278, 1983), oligonucleotide ligationassays (OLAs) (Landegren et al., Science 241:1077, 1988), and the like.Molecular techniques for DNA analysis have been reviewed (Landegren etal., Science 242:229-237, 1988).

In one specific, non-limiting example, the agent is an antagonist for areceptor on an NPY neuron, or a POMC neuron. Thus, the agent can be, butis not limited to, an antagonist of a Y2 receptor. Anelectrophysiological property of the POMC neurons is measured. Increasedactivity of NPY neurons, measured as increased frequency of IPSCs inPOMC neurons, hyperpolarization of POMC neurons, and/or a decrease inthe action potential firing rate of POMC neurons indicates theantagonist is of use in increasing feeding behavior. Without being boundby theory, antagonists, such as Y2 antagonists, can stimulate NPYneurons by reducing the tonic inhibition of those neurons mediated bythe Y2 R and as such will be of use in treating anorexia and cachexia.Thus, the methods described herein can be use to screen for agents thatincrease appetite, food intake, caloric intake and decrease energyexpenditure.

In another specific, non-limiting example the agent is an agonist for aleptin receptor on a POMC neuron or an NPY neuron that makes synapticcontact with a POMC neuron. An electrophysiological property of the POMCneuron is measured. Increased activity of POMC neurons, measured asdecreased frequency of IPSCs in POMC neurons, depolarization of POMCneurons, and/or an increase in the action potential firing rate of POMCneurons indicates the agonist is of use in decreasing feeding behavior.

The binding affinities of receptor agonists (or antagonists) can also bedetermined in either cells or a membrane preparation expressing thereceptor. For example, assays are utilized in which a labeled ligand isemployed. A number of labels have been indicated previously (e.g.,radiolabels, fluorescence labels, among others) to be of use. Thecandidate compound is added in an appropriate buffered medium. After anincubation to ensure that binding has occurred, the surface may bewashed free of any nonspecifically bound components of the assay medium,particularly any nonspecifically bound labeled ligand, and any labelbound to the surface determined. The label may be quantitativelymeasured. By using standards, the relative binding affinity of acandidate compound can be determined.

Following screening using the methods disclosed herein, further testingcan be performed, either in animal models or in clinical trials, toconfirm that the agent affects food intake, caloric intake, appetite, orenergy expenditure. Similarly further testing can be done to confirmthat the agent affects body temperature, blood pressure, stress-inducedanalgesia, reproductive function, cognitive abilities, emotional states,rewarding behavior, responses to drugs of abuse, including opioids andethanol, and neuroimmunomodulation. Exemplary in vivo assays for foodintake, caloric intake, appetite, or energy expenditure are described inthe Examples section below. However, one of skill in the art can readilydesign alternative in vivo assays or clinical trials.

A PYY agonist or antagonist can be screened using the methods disclosedherein, in order to determine if the PYY agonist will affect caloricintake, food intake, appetite, and/or energy metabolism. A PYY agonistis a molecule that binds to a receptor that specifically binds PYY, andelicits an effect of PYY. Suitable PYY agonists and antagonists that canbe screened using the methods disclosed herein include compounds thatbind specifically in a Y receptor assay or competes for binding withPYY, such as in a competitive binding assay with labeled PYY. SuitablePYY agonists include, but are not limited to, compounds that bind to theY2 receptor.

Thus, in one embodiment, a PYY agonist is selected using the methodsdisclosed herein that binds to a NPY neuron in the arcuate nucleus, andresults in an electrophysiological effect on an NPY neuron. Theelectrophysiological effect on the NPY neuron can result in a furtherelectrophysiological effect on a POMC neuron. Thus, one specific,non-limiting example, a PYY agonist is selected, using the methodsdisclosed herein, that causes depolarization of the membrane potentialof a POMC neuron. In another specific, non-limiting example, a PYYagonist is selected using the method disclosed herein that causes andecrease in IPSCs in a POMC neuron, and/or an increased activity of aPOMC neuron. In several non-limiting examples, agonists that causehyperpolarization of the membrane potential of a POMC neuron, increasein IPSCs in a POMC neuron, are selected using the methods disclosedherein.

PYY and agonists that can be screened using the methods disclosed hereininclude, but are not limited to, polypeptides comprising, oralternatively consisting of, the amino acid sequence for PPY andagonists thereof, e.g., mutants, fragments and/or variants thereof.Variants include deletions, insertions, inversions, repeats andsubstitutions (e.g., conservative substitutions and non-conservativesubstitutions). More than one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, etc.) can be deleted or inserted or substituted with another aminoacid. Typically conservative substitutions are the replacements, one foranother, among the aliphatic amino acids Ala, Val, Leu and Ile;interchange of Ser and Thr containing hydroxy residues, interchange ofthe acidic residues Asp and Glu, interchange between the amide residuesAsn and Gln, interchange of the basic residues Lys and Arg, interchangeof the aromatic residues Phe and Tyr, and interchange of the small-sizedamino acids Ala, Ser, Thr, Met and Gly.

As another example, polypeptide fragments may contain a continuousseries of deleted residues from the amino (N)- or the carboxyl(C)-terminus, or both. Any number of amino acids, ranging from 1 to 24,can be deleted from the N-terminus, the C-terminus or both.

Furthermore, the agonist polypeptides that are screened using themethods disclosed herein, also include, but are not limited to,polypeptides comprising, or alternatively consisting of, internaldeletions of the amino acid sequences for PPY and/or agonist thereof.Such deletions may comprise one or more amino acid residue deletions(e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.)and may begin at any amino acid position (e.g., two, three, four, five,six, seven, eight, nine, ten, etc.). In addition, polypeptides can bescreened that contain one or more such internal deletions. Suchdeletions are can be made in PPY, NPY and PP.

Also contemplated is the screening of agonist peptides that are PPY, NPYand/or PP chimeras having high affinity and/or selectivity for the Y2receptor. These chimeras may comprise amino acid substitutions of one ormore amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) from PPY,NPY and/or PP, variants, mutants and/or deletions thereof, with one ormore amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) from asecond PPY, NPY, or PP, variants, mutations and/or deletions thereof.These substitutions may begin at any amino acid position (e.g., two,three, four, five, six, seven, eight, nine, ten, etc.).

In one embodiment, the agents that are screened using the methodsdisclosed herein are selective for the Y2 receptor. That is, they bindwith higher affinity to Y2 compared to other receptors, such as Y1, Y2,Y3, Y4, Y5 and Y6. In another embodiment, the peptides are selective forthe Y2 and Y5 receptors over the Y1, Y3, Y4 and Y6 receptors.

Other polypeptide fragments that can be screened are fragmentscomprising structural or functional domain of the polypeptides of thisdisclosure. Such fragments include amino acid residues that comprise apolyproline-type II helix (residues 1-8), beta-turn (residues 9-14),amphipathic alpha-helix (residues 15-32) and/or a C-terminal turnstructure (residues 33-36). See, Kirby et al., J Med Chem 36:385-393,1993.

In addition, this disclosure includes the screening of a polypeptide oragonist comprising, or alternatively consisting of, the amino acidsequence for PPY, NPY and PP species variants and/or mutants, andfragments thereof. Also contemplated is the screening of fusionproteins, whereby a PYY or PYY agonist will be fused to another proteinor polypeptide (the fusion partner) using recombinant methods known inthe art, to identify fusion proteins of use in reducing appetite,caloric intake, food intake, and/or energy expenditure. These fusionproteins can be synthetically synthesized by any known method. Any knownpeptide or protein can be used as the fusion partner (e.g., serumalbumin, carbonic anhydrase, glutathione-S-transferase or thioredoxin,etc.).

The disclosure is illustrated by the following non-limiting Examples:

EXAMPLES Example 1 Material and Methods

Generation of POMC-EGFP mice: The EGFP cassette contains its own Kozakconsensus translation initiation site along with SV40 polyadenylationsignals downstream of the EGFP coding sequences directing properprocessing of the 3′ end of the EGFP mRNA. The EGFP cassette wasintroduced by standard techniques into the 5′ untranslated region ofexon 2 of a mouse Pomc genomic clone containing 13 kb of 5′ and 2 kb of3′ flanking sequences (Young et al., J Neurosci 18:6631-40, 1998). Thetransgene was microinjected into pronuclei of one-cell stage embryos ofC57BL/6J mice (Jackson Laboratories) as described (Young et al., JNeurosci 18:6631-40, 1998). One founder was generated and bred towildtype C57BL/6J to produce N₁ hemizygous mice. In addition, N₂ andsubsequent generations of mice homozygous for the transgene were alsogenerated. The mice are fertile and have normal growth and development.

Immunofluorescence and GFP co-localization: Anesthetized mice wereperfused transcardially with 4% paraformaldehyde and free-floating brainsections prepared with a vibratome. Sections were processed forimmunofluorescence and colocalization of GFP fluorescence using standardtechniques. Primary antisera and their final dilutions were rabbitanti-β-endorphin, 1:2500 v/v; rabbit anti-NPY, 1:25,000 v/v (AlanexCorp.); rabbit anti-ACTH, 1:2000 v/v; rabbit anti-GFP 1:10,000 v/v(AbCam); and mouse anti-TH, 1:1000 v/v (Incstar). After rinsing,sections were incubated with 10 mg/ml biotinylated horseanti-mouse/rabbit IgG (Vector Laboratories) followed by Cy-3 conjugatedstreptavidin, 1:500 v/v (Jackson Immunoresearch Laboratories).Photomicrographs were taken on a Zeiss Axioscop using FITC and RITCfilter sets (Chroma Technology Corp.). Other tissue sections weredeveloped with ABC reagent (Vector Laboratories) and eitherdiaminobenzidine or benzidine dihydrochloride as the chromagen.

Electrophysiology (Example 2): 200 μm thick coronal slices were cut fromthe ARC of four-week old male POMC-EGFP mice. Slices were maintained in(in mM) [NaCl, 126; KCl, 2.5; MgCl₂, 1.2; CaCl₂.2H₂O, 2.4; NaH₂PO₄.H₂O,1.2; NaHCO₃, 21.4; Glucose, 11.1] (Krebs) at 35° C. and saturated with95% O₂ 5% CO₂ for 1 hour (hr) prior to recordings. Recordings were madein Krebs at 35° C. Slices were visualized on an Axioskop FS2 (Zeiss)through standard infra red optics and using epifluorescence through aFITC filter set (see FIG. 1 c). Whole cell recordings were made fromfluorescent neurons using an Axopatch 1D amplifier (Axon Instruments)and Clampex 7 (Axon Instruments). Resting membrane potentials weredetermined using an event detection protocol on a PowerLab system (ADInstruments, Mountain View, Calif.) to average expanded traces of themembrane potential. Drugs were applied to the bath over the timesindicated. The resting membrane potential was stable for up to an hourin cells treated with Krebs alone. I-V relationships for the Met-Enkcurrents were established using a step protocol; (−60 mV holdingpotential, sequentially pulsed (40 ms) from −120 to −50 mV, cells werereturned to −60 mV for 2 seconds between voltage steps). The protocolwas repeated after Met Enk addition. The net current was the differencebetween the two I-V relationships. This protocol was repeated in Krebswith 6.5 mM K⁺. I-V relationships to identify the postsynaptic leptincurrent were performed similarly with slow voltage ramps (5 mV/s from−100 to −20 mV) before and 10 minutes after the addition of leptin (100nM). GABAergic IPSCs were recorded using a CsCl internal electrodesolution (in mM) [CsCl, 140; Hepes, 10; MgCl₂, 5; Bapta, 1; (Mg)-ATP, 5;(Na)GTP, 0.3]. Both mini IPSCs and large amplitude (presumablymultisynaptic) IPSCs were observed in the untreated slices. TTX (1 μM)abolished large IPSCs. Data were acquired before and after addition ofdrug for the times indicated on the figures at a −50 mV holdingpotential in 2 second sweeps every 4 seconds Mini postsynaptic currentswere analyzed using Axograph 4 (Axon Instruments). IPSCs and excitatorypostsynaptic currents (EPSCs) were distinguished on the basis of theirdecay constants; additionally picrotoxin (100 μM) blocked all IPSCs.POMC neurons receive a low EPSC tone and the frequency was not modulatedby any of the treatments described here.

Immunostaining for light and electron microscopy: Doubleimmunocytochemistry for NPY and POMC using different colordiaminobenzidine(DAB) chromogens was carried out on fixed mousehypothalami according to published protocols (Horvath et al.,Neuroscience 51:391-9, 1992). For electron microscopy, preembeddingimmunostaining for β-endorphin was using an ABC Elite kit (VectorLaboratories) and a DAB reaction followed by post-embedding labeling ofGABA and NPY using rabbit anti-GABA, 1:1000 v/v and gold conjugated (10nm) goat anti-rabbit IgG or sheep anti-NPY and gold conjugated (25 nm)goat anti-sheep IgG. Finally, sections were contrasted with saturateduranyl acetate (10 minutes) and lead citrate (20-30 seconds) andexamined using a Philips CM-10 electron microscope.

Animals: Male Pomc-EGFP mice were studied at 5-6 weeks of age and weregenerated as described above. Male mice aged 8-12 weeks and between20-30 g bodyweight were kept under controlled temperature (21-23° C.)and light conditions (lights on 06:00-18:00) with ad libitum access towater and food except where stated. All studies were performed in theearly light-phase (0700-0800).

Intraperitoneal injections: Mice were accustomed to IP injection byinjections of 0.5 ml saline on the two days prior to study. For allstudies, animals received an IP injection of either PYY₃₋₃₆ or saline in100 μl.

Electrophysiology: Whole cell patch clamp recordings were made from POMCneurons in the hypothalamus of 180 μm thick coronal slices fromPomc-EGFP mice, as previously reported (Cowley et al., Nature411:480-484, 2001). “Loose cell-attached” recordings were made usingextracellular buffer in the electrode solution, and maintaining sealresistance between 3-5 Mohm throughout the recording. Firing rates wereanalyzed using mini-analysis protocols (MiniAnalysis, Jaejin Software,N.J.). Vehicle controls were used in this system, previously validatedfor the electrophysiological actions of neuropeptides (Cowley et al.,Nature 411:480-484, 2001). Data were analyzed by ANOVA, Neuman-Keulsposthoc comparison, and Wilcoxon Signed Rank Test.

C-fos expression: C-fos expression was measured in Pomc-EGFP mice 2hours after IP administration of saline or PYY₃₋₃₆ (5 μg/100 g) usingstandard immunohistochemical techniques (Hoffinan et al., Front.Neuroendocrinol. 14:173-213, 1993). Data were obtained from 5 mice ineach group. For the Pomc-EGFP mice 5 anatomically matched arcuatenucleus sections (Franklin et al., The Mouse Brain in StereotaxicCoordinates (Academic Press, San Diego, 1997) were counted from eachanimal, and images acquired using a Leica TSC confocal microscope (Groveet al., Neuroscience 100:731-40, 2000).

Measurements of Energy Expenditure: To determine the actions of PYY onenergy expenditure the OXYMAX system (Columbus Instruments, Columbus,Ohio) is utilized with rodents following PYY injection into a treatmentcohort. This system is also utilized with rodents following a salineinjection (control cohort). The equipment measures O₂ consumption andCO₂ production; the efficiency with which the body produces CO₂ from O₂gives a reliable index of caloric or metabolic efficiency.

POMC sequences: A human large genomic contig (Homo sapiens chromosome 2reference contig) including the complete POMC gene and the nPOMC1,nPOMC2 and nPOMC3 elements can be found in GenBank, the NIH sequencedatabase maintained by the National Center for Biotechnology Information(NCBI). The information relating to this sequence is as follows:

-   -   Accession No.: NT_(—)005204.

A mouse large genomic contig (Mus musculus chromosome 12 WGSsupercontig) including the complete POMC gene and the nPOMC I, POMC2 andnPOMC3 elements) can also be found in GenBank. The information relatingto this sequence is as follows:

-   -   Accession No.: NW_(—)000041

A rat large genomic contig (Rattus norvegicus chromosome 6 WGSsupercontig) including the complete POMC gene and the nPOMC1, POMC2 andnPOMC3 elements) can also be found in GenBank. The information relatingto this sequence is as follows:

-   -   Accession NO.: NW_(—)043940

All these GenBank entries are incorporated herein by reference in theirentirety.

Example 2 Neural Network in the Arcuate Nucleus

A strain of transgenic mice was generated expressing green fluorescentprotein (EGFP Clontech), under the transcriptional control of mouse Pomcgenomic sequences that include a region located between −13 kb and −2 kbrequired for accurate neuronal expression (Young et al., J Neurosci18:6631-40, 1998) (FIG. 1 a). Bright green fluorescence (509 nm) wasseen in the two CNS regions where POMC is produced: the ARC and thenucleus of the solitary tract. Under ultraviolet (450-480 nm) excitationPOMC neurons were clearly distinguished from adjacent, non-fluorescentneurons (FIG. 1 b) visualized under infrared optics. Doubleimmunofluorescence revealed >99% cellular co-localization of EGFP andPOMC peptides within the ARC (FIG. 1 c). There was close apposition ofboth tyrosine hydroxylase (TH)- and NPY-stained terminals onEGFP-expressing POMC neurons, but no evidence of co-localization of theTH or NPY immunoreactivity with EGFP. Total fluorescent cell countsperformed on coronal hypothalamic sections revealed 3148±62 (mean±s.e.m.n=3) POMC-EGFP neurons distributed through the entire ARC (Franklin etal., The Mouse Brain in Stereotaxic Coordinates (Academic Press, SanDiego, Calif., 1997) (FIG. 1 d). POMC neurons in the mouse are locatedboth medially and ventrally within the ARC, in contrast to apredominantly lateral position in the rat ARC. FIG. 1 e showsfluorescently labeled neurons in the nucleus of the tractus solitarius.FIGS. 1 f-h show immunohistochemically labeled neurons in the nucleus ofthe solitary tract and area postrema. FIGS. 1 i-k show fluorescentlylabeled neurons in the granular cell layer of the dentate gyrus of thehippocampus. FIGS. 1 l-m show immunohistochemically labeled neurons inthe granular cell layer of the dentate gyrus of a young (two-month old)and an old (18-month old) POMC-EGFP mouse.

POMC-EGFP neurons in hypothalamic slices had a resting membranepotential of −40 to −45 mV and exhibited frequent spontaneous actionpotentials. The non-selective opioid agonist met-enkephalin (Met-Enk: 30μM; Sigma) caused a rapid (35-40 s), reversible hyperpolarization (10-20mV) of the membrane potential of POMC cells (n=10) and preventedspontaneous action potential generation (FIG. 2 a). In normal (2.5 mMK⁺) Krebs buffer, the reversal-potential of the inwardly-rectifyingopioid current was approximately −90 mV, while in 6.5 mM K⁺ Krebs thereversal-potential was shifted to approximately −60 mV (n=3: FIG. 2 b).The μ opioid receptor (MOP-R) antagonist CTAP (1 μM; PhoenixPharmaceuticals) completely prevented the current induced by Met-Enk inPOMC cells (n=3: FIG. 2 c). These characteristics indicate the opioidcurrent was due to activation of MOP-R and increased ion conductancethrough G protein coupled, inwardly-rectifying potassium channels (GIRK)(Kelly et al., Neuroendocrinology 52:268-75, 1990). The similar opioidresponses in EGFP-labeled POMC neurons to that of guinea pig (Kelly etal., Neuroendocrinology 52:268-75, 1990) or mouse (Slugg et al.,Neuroendocrinology 72:208-17, 2000). POMC cells, identified bypost-recording immunohistochemistry, suggests that expression of theEGFP transgene does not compromise either expression of receptors northeir coupling to second messenger systems in POMC neurons.

Next, the direct effects of leptin on identified POMC cells in slicepreparations were investigated. Leptin (0.1-100 nM) depolarized 72 of 77POMC cells by 3-30 mV (FIG. 3 a; mean±s.e.m. depolarization at 100 nMleptin=9.7±1.2 mV, n=45) within 2-10 minutes, in a concentrationresponsive manner (FIG. 3 b). There were two components to thedepolarization and neither were fully reversible within 40 minutes.Firstly, the depolarization was due to a small inward current whichreversed at approximately −20 mV (FIG. 3 c), suggesting the involvementof a non-specific cation channel (Powis et al., Am J Physiol 274,R1468-72, 1998). Secondly, leptin treatment decreased the GABAergic toneonto POMC cells. GABAergic inhibitory postsynaptic currents (IPSCs) wereobserved in POMC cells and leptin (100 nM) decreased their frequency by25% (FIG. 3 d) in 5 out of 15 cells suggesting that it actedpresynaptically to reduce GABA release (leptin had no effect on IPSCs in10 out of 15 POMC neurons). The effect on IPSC frequency occurred with asimilar lag to the effect on membrane potential. Thus, leptin not onlydirectly depolarizes POMC neurons but also acts at GABAergic nerveterminals to reduce the release of GABA onto POMC neurons, allowing themto adopt a more depolarized resting potential. The consistentdepolarization of POMC cells by leptin was specific because leptin hadno effect on 5 of 13 adjacent non-fluorescent cells tested (FIG. 3 e),while it hyperpolarized 5 (FIG. 3 f) and depolarized 3 other non-POMCneurons in the ARC. The electrophysiological effects of leptin reportedhere are consistent with leptin's biological actions; leptin rapidlycauses release of α-MSH from rat hypothalami (Kim et al., J Clin Invest105:1005-11, 2000), presumably by activating POMC neurons.

Previous reports of neuronal hyperpolarization by leptin (Glaum et al.,Mol Pharmacol 50:230-5, 1996; Spanswick et al., Nature 390:521-5, 1997),and the demonstrated co-localization of GABA and NPY (Horvath et al.,Brain Res 756:283-6, 1997) within subpopulations of ARC neurons,suggested that leptin hyperpolarizes NPY/GABA cells that directlyinnervate POMC neurons, and thus reduces GABAergic drive onto POMCcells. Both the leptin and NPY Y2 receptors are expressed on NPY neuronsin the ARC (Hakansson et al., J Neurosci 18:559-72, 1998; Broberger etal., Neuroendocrinology 66:393-408, 1997). Furthermore, activation of Y2receptors inhibits NPY release from NPY neurons (King et al., JNeurochem 73:641-6, 1999), and presumably would also diminish GABArelease from NPY/GABA terminals. This provides an alternativepharmacological approach, independent of leptin, to test thehypothesized innervation of POMC neurons by GABAergic NPY neurons.Indeed, NPY (100 nM; Bachem) decreased the frequency of GABAergic IPSCsby 55% within 3 minutes, in all 12 POMC cells tested (FIG. 4 a). BothNPY and leptin still inhibited IPSCs in the presence of tetrodotoxin(TTX) (6 of 6 and 3 of 5 cells respectively), indicating that some ofthe inhibition of IPSCs was occurring through direct effects atpresynaptic nerve terminals. POMC neurons express the NPY Y1 receptor(Broberger et al., Neuroendocrinology 66:393-408, 1997) and NPY alsohyperpolarized all POMC neurons tested, by an average of 9±6 mV (n=3).

Another pharmacological test to confirm the origin of GABAergicinnervation on POMC neurons from NPY/GABA terminals was to test theeffect of the recently characterized and highly selective MC3-R agonistD-Trp⁸-γMSH (Grieco et al., J Med Chem 43:4998-5002, 2000) on local GABArelease. D-Trp⁸-γMSH (7 nM) increased the frequency of GABAergic IPSCs(280±90%) recorded from 3 of 4 POMC neurons (FIG. 4 b). It had no effecton one cell. The positive effect of MC3-R activation, together with thenegative effects of NPY and leptin, demonstrate the dynamic range of theNPY/GABA synapse onto POMC neurons and point to the important role ofthis synapse in modulating signal flow within the ARC. D-Trp⁸-γMSH (7nM) also hyperpolarized (−5.5±2.4 mV) 9 of 15 POMC neurons tested anddecreased the frequency of action potentials (FIG. 4 c); the remainingcells showed no significant response to D-Trp⁸-γMSH. These effects couldbe due entirely to increased GABA release onto the POMC cells, or couldbe due to an additional postsynaptic action of D-Trp⁸-γMSH on POMCneurons, approximately half of which also express the MC3-R (Bagnol etal., J Neurosci (Online) 19, RC26, 1999). Thus, MC3-R acts in a similarautoreceptor manner to MOP-Rs on POMC neurons, diminishing POMC neuronalactivity in response to elevated POMC peptides.

To further determine that the IPSCs in POMC neurons were due to localinnervation by NPY/GABA cells, multi-label immunohistochemistry wasperformed using light and electron microscopy. Although independent NPY(Csiffary et al., Brain Res 506:215-22, 1990) and GABA (Horvath et al.,Neuroscience 51:391-9, 1992) innervation of POMC cells has beenreported, co-localization of NPY and GABA in nerve terminals formingsynapses onto POMC cells has not been shown. Similar to the rat(Csiffary et al., Brain Res 506:215-22, 1990), a dense innervation ofPOMC cells by NPY axon terminals was detected in the mouse (FIG. 4 d).Electron microscopy confirmed the coexpression of NPY and GABA in axonterminals and revealed that these boutons established synapses on theperikarya of all 15 ARC POMC neurons analyzed (representative example,FIG. 4 e).

A detailed model of regulation of this circuit shows dual mechanisms ofleptin action in the ARC, interactions between NPY/GABA and POMCneurons, and autoregulatory feedback from opioid and melanocortinpeptides as well as NPY (FIG. 4 f). In this model, leptin directlydepolarizes the POMC neurons and simultaneously hyperpolarizes thesomata of NPY/GABA neurons, and diminishes release from NPY/GABAterminals. This diminished GABA release disinhibits the POMC neurons,and result in an activation of POMC neurons and an increased frequencyof action potentials.

The ability to direct expression of a transgene to POMC neurons allowsmeasurement of the effects of agents on the interaction of NPY and POMCneurons. The effect of PYY on feeding in rats, and mice has beenestablished (Batterham et al., Nature 418:450, 2002). The effect of PYYon feeding in humans has been established (Batterham et al., Nature418:450, 2002. Thus, the ability to specifically detect POMC neurons,and to measure the effects of agents on the NPY/POMC circuit, allowsidentification of agents that affect caloric intake, food intake, andappetite.

Example 3 PYY Administration Affects c-fos Expression

To investigate whether this inhibition of food intake involved ahypothalamic pathway, c-fos expression was examined in the arcuatenucleus, an important center of feeding control (Schwartz et al., Nature404:661-671, 2000; Cowley et al., Nature 411:480-484, 2001), following asingle IP injection of PYY₃₋₃₆. There was a 2-fold increase in thenumber of cells positive for c-fos in the lateral arcuate of the rat(PYY₃₋₃₆=168±2, saline=82.7±5, n=3, P<0.0001). Likewise inPomc-EGFP-transgenic mice (Cowley et al., Nature 411:480-484, 2001) IPadministration of PYY₃₋₃₆ resulted in a 1.8-fold increase in the numberof arcuate cells positive for c-fos (FIG. 5 b), compared with salinecontrol animals (FIG. 5 a) (PYY₃₋₃₆=250±40, saline=137±15, n=5, P<0.05).IP PYY₃₋₃₆ caused a 2.6 fold increase in the proportion of POMC neuronsthat express c-fos (PYY₃₋₃₆=20.4±2.9%, saline=8±1.4%, n=5, P<0.006)(FIGS. 5 c and d). These observations show that PYY₃₋₃₆ can act via thearcuate nucleus

Example 4 Y2 Receptors

The electrophysiological response of hypothalamic POMC neurons toadministration of both PYY₃₋₃₆ and Y2A was examined. The effect of PYYon feeding in rats and mice has been established (Batterham et al.,Nature 418:450, 2002). POMC neurons were identified using mice withtargeted expression of green fluorescent protein in POMC neurons (Cowleyet al., Nature 411:480-484, 2001). PYY₃₋₃₆ disinhibited the POMCneurons, resulting in a significant depolarization of 19 of the 22 POMCneurons tested (FIG. 5 a inset) (10.3±2.1 mV depolarization, n=22,P<0.0003). A similar depolarization was seen with Y2A (8.7±1.8 mVdepolarization, n=9, P<0.002). The depolarization caused by PYY₃₋₃₆stimulated a significant increase in the frequency of action potentialsin POMC neurons (FIG. 6 a) (93% increase over control, P<0.05, n=22). Inthe whole cell mode the effect of PYY₃₋₃₆ was sometimes reversed uponwashout, but only after a long latency (30 minutes). A similar washoutof leptin effects upon these neurons was observed.

To exclude effects of cellular rundown, or seal deterioration, theeffects of PYY₃₋₃₆ in the “loose cell-attached” (or extracellular)configuration was examined. PYY₃₋₃₆ caused a reversible 5-fold increasein the frequency of action potentials in loose cell-attached recordingsof POMC neurons (FIG. 6 b). This increase in firing rate occurred withthe same latency as PYY₃₋₃₆ reduced the frequency of inhibitorypostsynaptic currents (IPSCs) onto all 13 POMC neurons tested (FIG. 6 c)(51.9±9.2% reduction, n=13, P<0.0001), indicating a reduced frequency ofGABA release onto POMC neurons. Interestingly, the firing rate of POMCneurons returned to basal, in spite of continued inhibition of IPSCs. Asimilar effect upon IPSC frequency was seen with Y2A (44.4±9.3%reduction, n=8, P<0.004) suggesting this effect to be via Y2R. PYY₃₋₃₆(25 nM) caused a hyperpolarization (5.2±1.16 mV, P<0.004, n=5) ofunidentified, but presumably NPY-containing, non-POMC, neurons in thearcuate nucleus. There is a tonic GABAergic inhibition of POMC neuronsby NPY neurons (Cowley et al., Nature 411:480-484, 2001) and theseresults suggest that PYY₃₋₃₆ acts by inhibiting NPY neurons, thusdecreasing this GABAergic tone and consequentially disinhibiting POMCneurons. The effect of Y2A on peptide secretion was also examined usinghypothalamic explants (Kim et al., J. Clin. Invest. 105:1005-11, 2000).Y2A significantly decreased NPY release, with a concomitant increase inα-MSH release from hypothalamic explants (Batterham et al., Nature418:450, 2002). Taken together these observations show that PYY₃₋₃₆modulates both the NPY and melanocortin systems in the arcuate nucleus.

Example 5 Analysis of the POMC Enhancer in Transgenic Mice

A strain of transgenic mice has been generated that expresses greenfluorescent protein under the transcriptional control of the mouse POMCgenomic sequences, including a region located between −13 kilobases (kb)and −2 kb that is required for accurate neuronal expression (see above,e.g. Example 2, and Cowley et al., Nature 411:480, 2001, which is hereinincorporated by reference). Additional lines of transgenic mice werethen generated. The starting material for these experiments was either a4 kb fragment of the rat POMC gene extending from a positionapproximately −4000 base pairs (bp) 5′ to the transcriptional start sitethrough to position +64 in the first exon or a 10 kb mouse genomic cloneincluding approximately 2 kb of 5′ flanking sequences. The complete POMCgene is composed of 3 exons and 2 introns, and approximately 2 kb of 3′flanking sequences (see Rubinstein et al., Neuroendocrinol. 58:373,1993, herein incorporated by reference).

A cosmid genomic library was constructed from 129S6 strain mouse genomicDNA partially cut with the EcoRI restriction endonuclease. This librarywas screened with probes generated from the original 10 kb mouse POMCclone. This screen resulted in the isolation of several overlapping POMCgenomic clones, which were used to construct a transgene vector formicroinjection that included approximately 13 kb of 5′ flankingsequences, the 6 kb POMC gene, and 8 kb of 3′ flanking sequences for atotal size of 27 kb. An artificial oligonucleotide sequence wasintroduced into exon 3 of the coding sequence to permit the unequivocalidentification and quantification of mRNA transcribed from the transgenecompared to the endogenous mouse POMC gene. Two additional transgeneswere constructed, one that was truncated at the −2 kb side 5′ to thePOMC gene and the other that was truncated at the +2 kb side 3′ to thePOMC gene (see Young et al., J. Neurosci. 18:6631, 1998, hereinincorporated by reference). Expression studies in this line oftransgenic mice demonstrated that DNA sequences between −13 and −2 kb 5′to the POMC gene are necessary for eutopic, neuron-specific expressionin the arcuate nucleus of the hypothalamus and the nucleus of thetractus solitarius. However, all the transgenes were appropriatelyexpressed in the pituitary gland, consistent with the location ofpituitary-specific DNA regulatory elements within the proximal −400 bpof the POMC promoter.

An additional series of transgenes containing the EGFP reporter genewere constructed as illustrated in FIG. 7. Truncation of the 5′ flankingsequences to a BamHI restriction site located at position −9 kb resultedin a loss of arcuate and NTS neuronal expression, but did not affectpituitary expression, suggesting that the essential arcuate and nucleusof the tractus solitarius (NTS) neuron-specific regulatory elements arecontained in the 4 kb between nucleotide positions −13 and −9 kb.Furthermore, an internal deletion of genomic 5′ flanking genomicsequences between positions approximately −6.5 to −0.7 or between −9 to0.7 kb did not affect the positive transgene expression in eitherhypothalamic neurons (see FIG. 8 a for a representative histologicsection illustrating the robust expression of the fluorescent protein inarcuate neurons) or the pituitary cells. This indicates the positionindependence of the neural regulatory elements relative to the basalpromoter. Virtually every transgenic strain produced with transgenesincluding the distal 4 kb of mouse POMC regulatory elements(approximately −13 to −9 kb from the transcriptional start site)displayed a high penetrance of reporter transgene expression in theneurons of the arcuate nucleus of the hypothalamus and the nucleus ofthe tractus solitrius, indicating that the transcriptional regulatoryelements contained within the 4 kb of DNA sequence are insulated fromthe effects of random chromosomal integration position in common withknown locus-control type enhancer elements.

Another transgene illustrated in FIG. 7 comprising the mouse POMCenhancer element from approximately −13 to −6.5 kb but with an internaldeletion of the sequences from approximately −6.5 to −0.7 kb directedeutopic transgene expression of a LacZ cassette encodingbeta-galactosidase to neurons of the arcuate nucleus, nucleus tractussolitarius, and corticotroph and melanotroph cells of the pituitarygland (not shown in a figure).

A transgene containing the distal 4 kb of mouse POMC 5′ genomicsequences between approximately −13 and −9 kb ligated to a minimalherpes simplex viral thymidine kinase (TK) promoter and human growthhormone reporter transgene (FIG. 7) was produced, and transgenic micecarrying this construct were generated. This region of the mouse POMCgenomic sequences conferred hypothalamic arcuate and NTSneuronal-specific expression of the human growth hormone marker (FIG. 8b).

The minimal TK promoter has been used previously in conjunction withproximal pituitary-specific POMC regulatory elements (see Liu, et al1992). In these studies, no intrinsic capacity of the TK promoter tocause reporter gene expression in POMC cells of transgenic mice wasobserved. Thus, expression of the hGH marker in these transgenic miceindicate that the 4 kb of distal mouse POMC genomic sequences contain aclassically defined position- and promoter-independent transcriptionalenhancer with specific activity for targeting high-level gene expressionto POMC neurons, in vivo.

To facilitate the generation of additional transgenic animals withexpression of marker genes or polypeptides from POMC gene regulatorysequences, a one-step POMC expression cassette was made (FIG. 7). Thisexpression cassette includes a polylinker with the nucleotide sequenceGCCCGGGCTCGAGTTTAAAGCGCGC (SEQ ID NO: 37) inserted into a StuIrestriction site present in the 5′ untranslated region of exon 2immediately upstream of the translational start codon for POMC. Thepolylinker comprises sequences for the uncommon SrfI, SmaI, XhoI, DraI,and BssHII restriction enzymes. Nucleotide sequences encoding a markeror polypeptide of interest can be ligated into one or more of theseuncommon restriction sites in a single cloning step. This is notpossible with the natural StuI site in this location, because numerousother StuI sites are present in the POMC gene. The position of thepolylinker in exon 2 is advantageous because it assures that foreign DNAsequences, with an appropriate Kozak consensus translational start site,ligated into this position will be translated into their encoded proteinbut the downstream POMC sequences will not be translated. The polylinkersequence can also be used as an oligonucleotide hybridization probe todemonstrate cell-specific expression of the transgene mRNA by in situhybridization histochemistry.

The POMC expression cassette comprising POMC enhancer elements locatedapproximately between −2 kb and the transcriptional start site conferstransgene expression specifically to the pituitary corticotrophs andmelanotrophs and the immature neurons of the granular cell layer of thedentate gyrus of the hippocampus (data not show in a figure). Theaddition of other POMC enhancer elements located between approximately−13 and −6.5 kb to the one-step expression cassette further conferstransgene expression to neurons of the arcuate nucleus and the nucleusof the solitary tract.

Example 6 Exemplary Screening Protocol

A number of lines of transgenic mice have been produced that carry atransgene including a POMC enhancer element operably linked to nucleicacid sequence encoding a marker. Histological sections can readily beprepared from these, or other lines of transgenic mice carrying a POMCregulatory region operably linked to a nucleic acid sequence encoding amarker. These sections can be used to screen agents, such as chemicalcompounds, proteins, small molecules or salts, in order to identify anagent that affects caloric intake, food intake, or appetite, asdescribed herein.

Coronal slices, from about 140 to about 400 μm thick, containing neuronsform the ARC of mice carrying a POMC gene or regulatory element operablylinked to a maker, wherein the marker is expressed in the POMC neuronsin the ARC. In one embodiment, suitable sections are produced from amale, four week old mouse expressing GFP from the POMC promoter, such asone of the lines of mice disclosed herein. It should be noted that theage and sex of the animal is not a limitation, as female mice as well asolder and younger mice can be used. These sections are then incubatedwith an agent of interest, and an electrophysiological parameter of thePOMC neurons is measured. A change in this electrophysiologicalparameter indicates that the agent affects caloric intake, food intake,appetite and/or energy expenditure.

In one example, 180 μm thick slices of the ARC are maintained in (in mM)[NaCl, 126; KCl, 2.5; MgCl₂, 1.2; CaC2₂H₂O, 2.4; NaH₂PO₄.H₂O, 1.2;NaHCO₃, 21.4; Glucose, 11.1] (Krebs) at 35° C. and saturated with 95% O₂5% CO₂ for 1 hour (hr) prior to recordings. Recordings are made in Krebsat 35° C. Slices are visualized on an Axioskop FS2 (Zeiss) throughstandard infra red optics and using epifluorescence through a FITCfilter set (see FIG. 1 c). Whole cell or loose cell attached recordingsare made from fluorescent neurons using an Axopatch 1D amplifier (AxonInstruments) and Clampex 7 (Axon Instruments).

Resting membrane potentials and action potential frequencies aredetermined using an event detection protocol on a PowerLab system (ADInstruments, Mountain View, Calif.) to average expanded traces of themembrane potential. Alternatively, an event detection software package,such as Synaptosoft (Gaegin software), is used to plot instantaneousfrequencies. Agents are applied to the bath over a specific time period,such as but not limited to, about one to about 15 minutes. The restingmembrane potential is stable for up to an hour in cells treated withKrebs alone.

Current to voltage (I-V) relationships are established using a stepprotocol; (−60 mV holding potential, sequentially pulsed (40 ms) from−120 to −50 mV, cells were returned to −60 mV for 2 s between voltagesteps). The protocol is repeated after addition of an agent of interest.The net current is the difference between the two I-V relationships areused to confirm that the agent is exerting a postsynaptic effect.Similarly slow voltage ramps (5 mV/s from −100 to −20 mV) before and 10minutes after the addition of the agent (such as, but not limited to, aconcentration of 1 nM-10 mM, e.g. at 100 nM) can be used to determine ifa postsynaptic effect is occurring.

GABAergic IPSCs are recorded using a CsCl internal electrode solution(in mM) [CsCl, 140; Hepes, 10; MgCl₂, 5; Bapta, 1; (Mg)-ATP, 5; (Na)GTP,0.3]. Addition of blockers of excitatory currents are used to allow theanalysis of IPSC frequency in isolation. Both mini IPSCs and largeamplitude (presumably multisynaptic) IPSCs are observed in the untreatedslices. TTX (1 mM) abolishes large IPSCs. Data is acquired before andafter addition of agent at, for example, a −50 mV holding potential in 2seconds sweeps every 4 seconds. Mini postsynaptic currents are analyzedusing Axograph 4 (Axon Instruments) or Synaptosoft. IPSCs and excitatorypostsynaptic currents (EPSCs) are distinguished on the basis of theirdecay constants and sensitivity to specific blocking agents; picrotoxin(100 mM) blocks IPSCs.

Exemplary parameters that can be analyzed are:

1. Analyzing membrane potential in POMC neurons as compared to acontrol, such as a POMC neuron in an untreated section or a sectionincubated with a control agent. This allows assessment of whether anagent increases or decreases the activity of POMC neurons.

2. Analyzing action potential firing rate in POMC neurons as compared toa control, such as a POMC neuron in an untreated section or a sectionincubated with a control agent. This allows assessment of whether anagent increases or decreases the activity of POMC neurons.

3. Analyzing IPSC frequency onto POMC neurons as compared to a control,such as an untreated section or a section incubated with a controlagent. This allows assessment of whether an agent increases or decreasesthe activity of NPY neurons.

Any change in one or more of these parameters identifies the agent asaffecting caloric intake, appetite, food intake, and/or energyexpenditure when a therapeutically effective amount of the agent isadministered to a subject. Thus, in one embodiment, these data areinterpreted by determining the effect of the agent on the activity ofPOMC neurons (as shown by membrane potential or action potential firingrate) and/or NPY/AGRP neurons (as shown by the IPSC frequency in POMCneurons). Agents that increase the activity of NPY neurons and/ordecrease the activity of POMC neurons will increase caloric intake, foodintake and/or appetite, and decrease energy expenditure. Agents whichdecrease the activity of NPY neurons and/or increase the activity ofPOMC neurons will decrease caloric intake, food intake and/or appetiteand/or increase energy expenditure.

Example 7 In Vitro Assessment of Ghrelin, a Known Appetite Stimulant

Slices are of the ARC from POMC-EGFP mice (see Example 1 for adescription of the animals) were maintained in (in mM) [NaCl, 126; KCl,2.5; MgCl₂, 1.2; CaCl₂.2H₂O, 2.4; NaH₂PO₄.H₂O, 1.2; NaHCO₃, 21.4;Glucose, 11.1] (Krebs) at 35° C. and saturated with 95% O₂ 5% CO₂ for 1hour (hr) prior to recordings. Recordings were made in Krebs at 35° C.Slices were visualized on an Axioskop FS2 (Zeiss) through standard infrared optics and using epifluorescence through a FITC filter set. Wholecell (FIG. 12 a and FIG. 12 b) or loose cell attached (FIG. 12 c)recordings were made from fluorescent neurons using an Axopatch 1Damplifier (Axon Instruments) and Clampex 7 (Axon Instruments).

GABAergic IPSCs were recorded using a CsCl internal electrode solution(in mM) [CsCl, 140; Hepes, 10; MgCl₂, 5; Bapta, 1; (Mg)-ATP, 5; (Na)GTP,0.3]. Addition of blockers of excitatory currents allowed the analysisof IPSC frequency in isolation. Both mini IPSCs and large amplitude(presumably multisynaptic) IPSCs were observed in the untreated slices.TTX (1 mM) abolished large IPSCs. Data was acquired before and afteraddition of agent at a −50 mV holding potential in 2 second sweeps every4 seconds. Picrotoxin (100 mM) blocked all IPSCs.

IPSC frequencies were analyzed using Synaptosoft (Gaegin software), todetermine instantaneous IPSC frequencies (FIG. 12 a). Resting membranepotentials are determined using an event detection protocol on aPowerLab system (AD Instruments, Mountain View, Calif.) to averageexpanded traces of the membrane potential (FIG. 12 b). Action potentialfiring rates were determined in loose cell attached mode and therecorded data was analyzed using Synaptosoft to determine instantaneousfrequencies. Ghrelin (50 nM) was applied to the bath over three minutes.The resting membrane potential was stable for up to an hour in cellstreated with Krebs alone.

The results of the addition of Ghrelin on IPSC frequency of POMC neuronsare shown in FIG. 12 a. An increase in the frequency of IPSCs in POMCneurons was detected. This is caused by NPY neurons. Thus an increase inthe activity of NPY neurons is demonstrated. FIG. 12 b shows the effectof Ghrelin on the resting membrane potential of POMC neurons. Ghrelinhyperpolarized POMC neurons, mean 1.47±0.7 mV; p<0.03. The activity ofPOMC neurons was decreased by the addition of Ghrelin. FIG. 12 c showsthe action potential firing rate in POMC neurons. The activity of POMCneurons was inhibited by Ghrelin. Thus, Ghrelin, an agent known toincrease feeding, caloric intake, and appetite, and decrease energyexpenditure, decreases the resting membrane potential (FIG. 12 b) andaction potential firing rate of POMC neurons (by 50%, see FIG. 12 c),increases the frequency of IPSCs in POMC neurons (FIG. 12 a), andincreases the activity of NPY neurons.

Example 8 In-Vitro Assessment of Fenfluramine, a Known AppetiteSuppressant and Weight Loss Agent

Slices are of the ARC from POMC-EGFP [the line that the may 2000 naturepaper describes] were maintained in (in mM) [NaCl, 126; KCl, 2.5; MgCl₂,1.2; CaCl₂.2H₂O, 2.4; NaH₂PO4.H₂O, 1.2; NaHCO₃, 21.4; Glucose, 11.1](Krebs) at 35° C. and saturated with 95% O₂ 5% CO₂ for 1 hour (hr) priorto recordings. Recordings were made in Krebs at 35° C. Slices werevisualized on an Axioskop FS2 plus (Zeiss) through standard infra redoptics and using epifluorescence through a endow-GFP filter set (ChromaTechnology Corp). Whole cell or loose cell attached recordings (bothwere used, results from whole cell recordings are shown in FIG. 13 b;results from loose cell attached are shown in FIG. 13 a) were made fromfluorescent neurons using an Axopatch 200B amplifier (Axon Instruments)and Clampex 8 (Axon Instruments).

Action potential firing rates were determined in loose cell attachedmode and the recorded data was analyzed using Synaptosoft to determineinstantaneous frequencies (FIG. 13 a). Resting membrane potentials anddepolarizations were determined using an event detection protocol on aPowerLab system (AD Instruments, Mountain View, Calif.) to averageexpanded traces of the membrane potential (FIG. 13 b). Serotoninreceptor agonists were applied to the bath at the specifiedconcentrations over four minutes. The resting membrane potential wasstable for up to an hour in cells treated with Krebs alone.

Fenfluramine (20 μM) caused a two fold increase in the frequency ofaction potentials in POMC neurons (FIG. 13 a; n=3). Thus fenfluramineincreased the activity of POMC neurons. In separate experimentsFenfluramine also depolarized POMC neurons (FIG. 13 b) in a dosedependent manner. Thus, by another test Fenfluramine increases theactivity of POMC neurons. The non-selective serotonin receptor agonistserotonin (5-HT) also increased the resting membrane potential(depolarized) POMC neurons. The effect of serotonin and fenfluramine onPOMC neurons is likely mediated by the 5-HT 2C R because 5-HT 2C Rselective agonists mCPP and MK 212 also depolarized POMC neurons (FIG.13 b).

Example 9 Sequences and the Identification of the nPOMC1, nPOMC2, andnPOMC3 Elements

The complete nucleotide sequence of the 4 kb of 129S6 substrain mousePOMC genomic DNA from approximately −13 kb to −9 kb relative to thetranscriptional start site was obtained from the cosmid clones. Amultiple alignment sequence comparison was performed with a public humandatabase BAC sequence containing the human POMC gene on chromosome 2using the web-based program PIP Maker (Percentage Identity Plot) and theprogram named Dotter. The two programs, which use a completely differentsequence comparison algorithm, found the same two highly conservedregions between mouse and human 5′ flanking POMC gene sequences (FIG.9). These two homology islands have been termed nPOMC1 and nPOMC2 forneural POMC regulatory elements 1 and 2.

FIGS. 10 a-10 c illustrate a sequence alignment of the nPOMC1 and nPOMC2elements from a variety of mammalian species. In addition to the primarysequence, the distance between both sites to the TATA box is alsoconserved in the human and mouse POMC genes. The nPOMC1 site extends forapproximately 450 bp with an overall mouse/human similarity of 65%having a 190 bp core with a maximal conservation of 80% (the human andbovine similarity is even higher, at 85%). This core is at −10.5 and−12.2 kb from the human and mouse TATA box, respectively. The core islocated 1.7 or 2.3 kb upstream of human and mouse nPOMC2, respectively.The site nPOMC2 has a 153 bp region from which 138 are identical betweenmouse and human (90% of similarity). It is located at −8.9 and −9.9 kbfrom the human and mouse TATA box, respectively.

Without being bound by theory, these two small and highly conservedareas, embedded within several kb of heterogeneous genomic sequences,resemble the exon-intron differences within the transcriptional unit.Interestingly, the similarities between mouse and human exon 1, 2 and 3of 64%, 87% and 82%, respectively, are not higher than the interspeciesidentity for nPOMC1 and nPOMC2 (FIGS. 11 a-11 b).

A Clustall comparison of 280 bp of the proximal human and mouse POMCpromoters show 68% of similarity in a region that contains allcis-acting elements necessary for basal (e.g. TATA and GC boxes) andpituitary specific expression (e.g. T-Pit, Ptx1 and PP1). Usingdegenerate oligonucleotide primers to amplify corresponding genomicregions from other mammalian species and sequencing of the PCRfragments, it was confirmed that nPOMC1 and nPOMC2 are also highlyconserved in bovine, hamster, and rabbit genomic DNA. In addition, abovine genomic library was screened using the bovine nPOMC1 PCR fragmentas a radiolabeled probe. One of the positive phage clones also containsnPOMC2 and POMC exon 1 indicating that these two regulatory regions aresyntenic with the TATA box within 15 kb, similar to the human and mouse.An internal portion of bovine nPOMC2 that was amplified from this cloneshows 90% similarity with the human.

The sequences of the rat nPOMC1 and nPOMC2 were obtained from a BLASTcomparison of the rat genome data base and they are highly similar tothe mouse sequence. The relative order of the two elements and therelative distances between themselves and between each element and thetranscriptional start site are also highly similar to both the mouse andthe human genes (FIGS. 14 a-14 g). Furthermore, the 129 mouse POMCgenomic sequence is nearly identical to the C57BL/6J POMC genomicsequence now available in GenBank. The nucleotide sequences spanning thenPOMC1, and nPOMC2 elements together with the precise nucleotidepositions on human chromosome 2, mouse chromosome 12, and rat chromosome6 are shown in FIG. 14. BLAST analyses indicate that the complete nPOMC1and nPOMC2 elements are previously unidentified and uncharacterizedsequences and appear to be unique to the POMC gene locus. Thus, one ofskill in the art can readily generate transgenic mice carrying atransgene including any of these regions of the POMC gene, operablylinked to a marker (such as, but not limited to, growth hormone or GFP).

The adipostatic hormone leptin not only activates POMC neurons but alsostimulates transcription of the POMC gene in the hypothalamus presumablythrough a JAK kinase/STAT3-dependent pathway. The web-based program MatInspector was used to localize STAT3 DNA binding sites within 11.5 kb of5′ flanking regions of the human POMC gene. Eight sites were detectedthat share high homology with the consensus core TTCCNGGAA (SEQ ID NO:35). Interestingly, only one site entirely matches this consensussequence and it is located within the highly conserved NPOMC1 site and50 bp downstream of another STAT3-like site (FIG. 10 b). This similaritysuggests that nPOMC1(3′ half) may be a leptin-responsive element withinthe POMC gene. The sites are slightly less well conserved in the othergenomic sequences available. Another potentially interesting short DNAsequence present in the 5′ half of nPOMC1 that is 100% identical amongall five mammalian species is CTAATGGATGTGCATTA (SEQ ID NO: 36).Excluding the 5° C., the remaining 16 nucleotides contain an imperfect(12/16) palindrome that could be a symmetrical DNA-protein binding site.

A BLAST comparison of mouse POMC genomic sequences between −9 and −0.8kb and human POMC genomic sequences between −7 and −0.8 kb revealed anadditional homology island of approximately 140 base pairs that wastermed nPOMC3. Further BLAST comparisons with the rat genome databasealso identified the same element in a corresponding region of the ratPOMC gene. The sequence of mouse, human, and rat nPOMC3 elements areshown in FIGS. 11 a-11 b with their exact nucleotide positions onchromosomes 12, 2, and 6 respectively. The sequence alignments fornPOMC3 among the three mammalian species is shown in FIG. 10 c. Thenucleotide sequences spanning the nPOMC3 elements together with theprecise nucleotide positions on human chromosome 2, mouse chromosome 12,and rat chromosome 6 are shown in FIGS. 14 a-14 g. BLAST analysesindicate that the complete nPOMC3 element is a previously unidentifiedand uncharacterized sequence and appears to be unique to the POMC genelocus. Thus, one of skill in the art can readily generate transgenicmice carrying a transgene including any of these regions of the POMCgene, operably linked to a marker (such as, but not limited to, growthhormone or GFP).

Additional percentage identities were calculated for nPOMC1, nPOMC2, andnPOMC3. The calculated percentage identities are as follows:

-   -   nPOMC1 (5′ half) (208 base pairs) 57% across 5 species or 65%        across 3 species (human, mouse, and rat)    -   nPOMC1 (3′ half) (326 base pairs) 69% across 3 species (human,        mouse, and rat)    -   nPOMC1 complete (534 base pairs) 67% across 3 species (human,        mouse, and rat)    -   nPOMC2 (234 base pairs) 75% across 5 species or 78% across 3        species (human, mouse, and rat)    -   nPOMC3 (145 base pairs) 78% across 3 species (human, mouse, and        rat)

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. An isolated proopiomelanocortin enhancer element, comprising thenucleic acid sequence set forth as SEQ ID NO: 9, wherein the isolatedproopiomelanocortin enhancer element elevates transcription of aheterologous coding sequence in a host cell, wherein theproopiomelanocortin element is isolated from the adjacent 5′ or 3′nucleic acid sequences of an endogenous chromosome comprising theproopiomelancortin gene.
 2. The isolated proopiomelanocortin enhancerelement of claim 1, consisting of the nucleic acid sequence set forth asSEQ ID NO:
 9. 3. The isolated proopiomelanocortin enhancer element ofclaim 1, comprising a 5′ half and a 3′ half, wherein the 5′ half and the3′ half have the nucleic acid sequence set forth, respectively, as a)SEQ ID NO: 1 and SEQ ID NO: 6; b) SEQ ID NO: 4 and SEQ ID NO: 7; or c)SEQ ID NO: 5 and SEQ ID NO:
 8. 4. The isolated proopiomelanocortinenhancer element of claim 1, comprising nucleic acid sequence as setforth as SEQ ID NO: 1 and the nucleic acid sequence set forth as SEQ IDNO:
 6. 5. The isolated proopiomelanocortin enhancer element of claim 1,further comprising a) an nPOMC2 element comprising the nucleic acidsequence as set forth as one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or b) an nPOMC3element comprising the nucleic acid sequence as set forth as one of SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19; or c) acombination thereof wherein the enhancer element directs expression of aheterologous nucleic acid sequence in a proopiomelanocortin neuron. 6.The isolated enhancer element of claim 1, comprising a nucleic acidsequence as set forth as SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22.7. The isolated enhancer element of claim 1, operably linked to apromoter and a heterologous nucleic acid sequence encoding apolypeptide, wherein the enhancer element directs expression of theheterologous nucleic acid sequence encoding the polypeptide in aproopiomelanocortin neuron.
 8. The isolated enhancer element of claim 7,wherein the polypeptide is an enzyme, a marker, a polypeptide thatconfers antibiotic resistance, or an antigen.
 9. The isolated enhancerelement of claim 7, wherein the polypeptide is green fluorescentprotein.
 10. The isolated enhancer element of claim 1, operably linkedto a heterologous promoter.
 11. An expression vector comprising theisolated enhancer element of claim 1 operably linked to a heterologouspromoter.
 12. The vector of claim 11, wherein the vector is a viralvector.
 13. The vector of claim 11, wherein the vector is a plasmidvector.
 14. An isolated host cell transformed with the vector of claim11.
 15. The isolated host cell of claim 14, wherein the host cell is aeukaryotic cell.
 16. The isolated host cell of claim 14, wherein thehost cell is a prokaryotic cell.
 17. An expression cassette comprisingthe POMC enhancer of claim 1, and a nucleic acid sequence encodingproopiomelanocortin comprising at least one exon, wherein a polylinkercomprising a unique site for a restriction enzyme is inserted into theexon.
 18. An isolated proopiomelanocortin enhancer element, comprisingthe nucleic acid sequence set forth as SEQ ID NO: 1, wherein theisolated proopiomelanocortin enhancer element elevates transcription ofa heterologous coding sequence in a host cell, wherein thepropiomelanocortin element is isolated from the adjacent 5′ or 3′nucleic acid sequences of an endogenous chromosome comprising theproopiomelanocortin gene.
 19. The isolated enhancer element of claim 18,operably linked to a promoter and a heterologous nucleic acid sequenceencoding a polypeptide, wherein the enhancer element directs expressionof the heterologous nucleic acid sequence encoding the polypeptide in aproopiomelanocortin neuron.
 20. The isolated enhancer element of claim19, wherein polypeptide is an enzyme, a marker, a polypeptide thatconfers antibiotic resistance, or an antigen.
 21. The isolated enhancerelement of claim 19, wherein the polypeptide is green fluorescentprotein.
 22. The isolated enhancer element of claim 19, operably linkedto a heterologous promoter.
 23. An expression vector comprising theisolated enhancer element of claim 19 operably linked to a heterologouspromoter.
 24. The vector of claim 23, wherein the vector is a viralvector.
 25. The vector of claim 23, wherein the vector is a plasmidvector.
 26. An isolated host cell transformed with the vector of claim23.
 27. The isolated host cell of claim 26, wherein the host cell is aeukaryotic cell.
 28. The isolated host cell of claim 27, wherein thehost cell is a prokaryotic cell.